A novel INS mutation in a family with maturity-onset diabetes of the young: Variable insulin secretion and putative mechanisms.

Insulin gene (INS) mutations cause a rare form of maturity-onset diabetes of the young (MODY), a heterogeneous group of autosomal dominant diabetes with at least 14 confirmed causative genes. Here, we describe a family with MODY due to a novel INS mutation, detected using massively parallel sequencing (MPS). The proband presented aged 11 years with mild diabetic ketoacidosis. She was negative for IA2 and GAD antibodies. She had a strong family history of diabetes affecting both her two siblings and her mother, none of whom had ketosis but who were considered to have type 1 diabetes and managed on insulin, and her maternal grandfather, who was managed for decades on sulfonylureas. Of note, her younger sister had insulin deficiency but an elevated fasting proinsulin:insulin ratio of 76% (ref 5%-30%). Sanger sequencing of HNF4A, HNF1A, and HNF1B in the proband was negative. Targeted MPS using a custom-designed amplicon panel sequenced on an Illumina MiSeq detected a heterozygous INS mutation c.277G>A (p.Glu93Lys). Sanger sequencing confirmed the variant segregated with diabetes within the family. Structural analysis of this variant suggested disruption of a critical hydrogen bond between insulin and the insulin receptor; however, the clinical picture in some individuals also suggested abnormal insulin processing and insulin deficiency. This family has a novel INS mutation and demonstrated variable insulin deficiency. MPS represents an efficient method of MODY diagnosis in families with rarer gene mutations.

Treatment of Lesch-Nyhan disease with S-adenosylmethionine: experience with five young Malaysians, including a girl.

BACKGROUND: Lesch-Nyhan disease (LND) is a rare X-linked recessive neurogenetic disorder caused by deficiency of the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT, EC 2.4.2.8) which is responsible for recycling purine bases into purine nucleotides. Affected individuals have hyperuricemia leading to gout and urolithiasis, accompanied by a characteristic severe neurobehavioural phenotype with compulsive self-mutilation, extrapyramidal motor disturbances and cognitive impairment. AIM: For its theoretical therapeutic potential to replenish the brain purine nucleotide pool, oral supplementation with S-adenosylmethionine (SAMe) was trialed in 5 Malaysian children with LND, comprising 4 related Malay children from 2 families, including an LND girl, and a Chinese Malaysian boy. RESULTS: Dramatic reductions of self-injury and aggressive behaviour, as well as a milder reduction of dystonia, were observed in all 5 patients. Other LND neurological symptoms did not improve during SAMe therapy. DISCUSSION: Molecular mechanisms proposed for LND neuropathology include GTP depletion in the brain leading to impaired dopamine synthesis, dysfunction of G-protein-mediated signal transduction, and defective developmental programming of dopamine neurons. The improvement of our LND patients on SAMe, particularly the hallmark self-injurious behaviour, echoed clinical progress reported with another purine nucleotide depletion disorder, Arts Syndrome, but contrasted lack of benefit with the purine disorder adenylosuccinate lyase deficiency. This first report of a trial of SAMe therapy in LND children showed remarkably encouraging results that warrant larger studies.

Early diagnosis of adenylosuccinate lyase deficiency using a high-throughput screening method and a trial of oral S-adenosyl-l-methionine as a treatment method.

AIM: The aim of this study was to develop a high-throughput urine screening technique for adenylosuccinate lyase (ADSL) deficiency and to evaluate S-adenosyl-l-methionine (SAMe) as a potential treatment for this disorder. METHOD: Testing for succinyladenosine (S-Ado), a marker of ADSL deficiency, was incorporated into a screening panel for urine biomarkers for inborn errors of metabolism using electrospray tandem mass spectrometry. Liquid chromatography-mass spectrometry and high-performance liquid chromatography were used to confirm and monitor the response of metabolites to oral SAMe treatment. RESULTS: Increased levels of S-Ado were detected in a 3-month-old male infant with hypotonia and seizures. ADSL gene sequencing revealed a previously described c.-49T>C mutation and a novel c.889_891dupAAT mutation, which was likely to disrupt enzyme function. After 9 months of SAMe treatment, there was no clear response evidenced in urine metabolite levels or clinical parameters. INTERPRETATION: These results demonstrate proof of the principle for the high-throughput urine screening technique, allowing earlier diagnosis of patients with ADSL deficiency. However, early treatment with SAMe does not appear to be effective in ADSL deficiency. It is suggested that although SAMe treatment may ameliorate purine nucleotide deficiency, it cannot correct metabolic syndromes in which a toxic nucleotide is present, in this case presumed to be succinylaminoimidazole carboxamide ribotide.

A novel NR5A1 variant in an infant with elevated testosterone from an Australasian cohort of 46,XY patients with disorders of sex development.

BACKGROUND: NR5A1 loss-of-function mutations are increasingly found to be the cause of 46,XY disorders of sex development (DSD). OBJECTIVE: To determine the presence of NR5A1 mutations in an Australasian cohort of 17 46,XY DSD patients with presumed androgen insensitivity syndrome (AIS) who were negative for androgen receptor gene (AR) mutation. DESIGN: Exons 2-7 of NR5A1 were PCR amplified and sequenced. Gene expression and cellular localization studies were performed on a novel NR5A1 variant c.74A>G (p.Y25C) identified in this study. RESULTS: We identified one novel mutation, c.74A>G (p.Y25C) in a patient characterized by penoscrotal hypospadias with bifid scrotum. He had elevated testosterone and gonadotropins in early infancy. Functional analysis of p.Y25C in vitro demonstrated reduced transcriptional activation by SF-1 and partially impaired nuclear localization in a proportion of transfected human adrenal NCI-H295R cells. CONCLUSION: This is the first reported case of a DSD patient with a NR5A1 mutation and elevated testosterone levels. Our finding supports evaluation of NR5A1 mutations in 46,XY DSD patients with a range of testosterone levels.

The placenta in Beckwith-Wiedemann syndrome: genotype-phenotype associations, excessive extravillous trophoblast and placental mesenchymal dysplasia.

AIMS: Placental mesenchymal dysplasia (PMD) is a rare condition which is associated with the disparate fetal outcomes of Beckwith-Wiedemann syndrome (BWS), fetal growth restriction or intrauterine and neonatal death. We aimed to investigate the potential epigenetic/genetic anomalies associated with PMD and their relationship with the different causes of BWS. METHODS: Eight archival cases in which PMD, BWS or both were diagnosed were investigated by correlating morphology with p57 Kip2 expression, XY fluorescence in situ hybridisation (FISH) analysis and DNA genotyping. RESULTS: Placentae from BWS cases caused by aberrant IC2 methylation, leading to abnormal p57 Kip2 expression, did not show PMD but had a striking excess of extravillous trophoblast. PMD in the absence of BWS was caused by androgenetic/biparental mosaicism. The single case of BWS with PMD was due to mosaic uniparental disomy of 11p15.5. In the latter two aetiologies, our results indicate that the uniparental disomy is confined to the villous mesenchyme. CONCLUSIONS: These results suggest that the link between PMD and BWS is uniparental disomy of genes confined to the telomeric IC1 region of 11p15.5. A strong candidate gene is IGF2, a known growth factor of placental mesenchyme.

Is it time to commence newborn screening for congenital adrenal hyperplasia in Australia?

21-Hydroxylase deficiency (21-OHD) is the most common cause of congenital adrenal hyperplasia, with an incidence of 1:14000 live births and equal prevalence among males and females. Newborns with the most severe "salt-wasting" form of 21-OHD are susceptible to salt-wasting crises in the first few weeks of life. This is associated with morbidity and mortality. 21-OHD newborn screening (NBS) is currently performed in many countries. Despite several prominent medical societies recommending 21-OHD NBS, no state in Australia currently screens for this condition. We report a case that illustrates the need to reconsider including 21-OHD in NBS. 21-OHD NBS can be reliable, sensitive and effective in reducing morbidity and mortality.

Adenylosuccinate lyase deficiency in a Malaysian patient, with novel adenylosuccinate lyase gene mutations.

Most cases of adenylosuccinate lyase (ADSL OMIM 103050) deficiency reported to date are confined to the various European ethnic groups. We report on the first Malaysian case of ADSL deficiency, which appears also to be the first reported Asian case. The case was diagnosed among a cohort of 450 patients with clinical features of psychomotor retardation, global developmental delay, seizures, microcephaly and/or autistic behaviour. The patient presented with frequent convulsions and severe myoclonic jerk within the first few days of life and severe psychomotor retardation. The high performance liquid chromatography (HPLC) profile of the urine revealed the characteristic biochemical markers of succinyladenosine (S-Ado) and succinyl-aminoimidazole carboximide riboside (SAICAr). The urinary S-Ado/SAICAr ratio was found to be 1.02 (type I ADSL deficiency). The patient was compound heterozygous for two novel mutations, c.445C > G (p.R149G) and c.774_778insG (p.A260GfsX24).

Investigating maturity onset diabetes of the young.

Maturity Onset Diabetes of Young (MODY) is a monogenic and autosomal dominant form of diabetes mellitus with onset of the disease often before 25 years of age. It is due to dysfunction of pancreatic beta cells characterised by non-ketotic diabetes and absence of pancreatic auto-antibodies. It is frequently mistaken for type 1 or type 2 diabetes mellitus. Diagnosis of MODY is important as the GCK subtype has better prognosis and may not require any treatment. Subtypes HNF1A and HNF4A are sensitive to sulfonylureas, however diabetes complications are common if not treated early. Moreover, there is genetic implication for the patient and family. Rare MODY subtypes can be associated with pancreatic and renal anomalies as well as exocrine dysfunction of the pancreas. So far there are six widely accepted subtypes of MODY described but the list has grown to nine. Although the majority of diabetes mellitus in youth remains type 1 and the incidence of type 2 is rising, MODY should be considered in patients with non-ketotic diabetes at presentation, and in patients with a strong family history of diabetes mellitus without pancreatic auto-antibodies. Furthermore the diagnosis must be confirmed by molecular studies. With advancement in genomic technology, rapid screening for MODY mutations will become readily available in the future.

The clinical and biochemical spectrum of congenital adrenal hyperplasia secondary to 21-hydroxylase deficiency.

21-Hydroxylase Deficiency (21-OH Deficiency) represents the most common form of Congenital Adrenal Hyperplasia (CAH), a complex and heterogenous group of conditions, characterised by defects in one of the five enzymes involved in adrenal steroidogenesis. Defects in this steroidogenic enzyme, the product of the CYP21A2 gene, cause disruption in the pathway involved in cortisol and aldosterone production and consequently, the accumulation of their steroid precursors as well as a resulting adrenocorticotrophic hormone (ACTH)-driven overproduction of adrenal androgens. Treatment with glucocorticoid, with or without mineralocorticoid and salt replacement, is directed at preventing adrenal crises and ensuring normal childhood growth by alleviating hyperandrogenism. Conventionally, two clinical forms of 21-OH Deficiency are described - the classical form, separated into salt-wasting and simple-virilising phenotypes, and the non-classical form. They are differentiated by their hormonal profile, predominant clinical features and age of presentation. A greater understanding of the genotype-phenotype correlation supports the view that 21-OH Deficiency is a continuum of phenotypes as opposed to a number of distinct phenotypical entities. Significant advancements in technologies such as Tandem Mass Spectrometry (TMS) and improvements in gene analysis, such as complete PCR-based sequencing of the involved gene, have resulted in remarkable developments in the areas of diagnosis, treatment and treatment monitoring, neonatal screening, prenatal diagnosis and prenatal therapy.

Utility of AVP gene testing in familial neurohypophyseal diabetes insipidus.

CONTEXT: Familial neurohypophyseal diabetes insipidus (FNDI) is a rare disorder resulting from arginine vasopressin (AVP) gene mutations. A partial defect in AVP secretion occurs early in the course of FNDI and may not be detected by a water deprivation test (WDT). Testing for AVP gene mutations may confirm a diagnosis of FNDI when a WDT is inconclusive and may also predict individuals who will later develop FNDI. OBJECTIVE: To test the utility of AVP gene analysis in confirming the diagnosis of FNDI. PATIENTS: Five families (20 subjects, 14 symptomatic and six asymptomatic) with FNDI and nine children with idiopathic neurohypophyseal diabetes insipidus (INDI). MEASUREMENTS: Genomic DNA was analysed for AVP gene mutations using polymerase chain reaction (PCR) amplification and sequencing. RESULTS: Heterozygous AVP gene mutations were found in all subjects with FNDI but none of the ICDI patients. Each family had their own distinct mutation. We identified two novel mutations (C44W and C105S). One asymptomatic subject developed diabetes insipidus (DI) 4 months after detection of an AVP gene mutation. The WDT suggested partial DI in 4/6 but was normal in 2/6 children with FNDI. CONCLUSION: AVP gene testing allowed diagnostic confirmation of FNDI when the WDT was inconclusive in symptomatic children, therefore obviating the need for a repeat WDT and enabling earlier initiation of appropriate treatment. AVP gene testing also has the potential to identify which asymptomatic children will later develop FNDI.

Autopsy diagnosis of 21-hydroxylase deficiency CAH in a case of apparent SIDS.

A 5-month-old boy with no history of vomiting, early sexual development, or noticeable significant illness was found dead in bed. Autopsy demonstrated bilateral adrenal hyperplasia unequivocally shown on biochemical testing of blood and urine to be due to 21-hydroxylase deficiency. Genetic analysis of the CYP21 gene showed compound heterozygosity; 1 allele contained a pseudogene sequence (gene conversion) and the other contained a previously described I172N point mutation. On theoretical grounds, the genotype would have been expected to cause simple virilizing congenital adrenal hyperplasia but, because no other cause of death could be found, it is possible that it caused a fatally severe loss of enzyme activity in this child. If this assumption is valid, newborn screening would have prevented this death, had it been available.

Two novel mutant human adenylosuccinate lyases (ASLs) associated with autism and characterization of the equivalent mutant Bacillus subtilis ASL.

An Australian patient with autism was found to be heterozygous for two mutations in the gene encoding adenylosuccinate lyase (ASL), resulting in the protein mutations E80D and D87E. The patient's mother carried only the E80D mutation. The equivalent positions are 62 and 69 in Bacillus subtilis ASL. Although both human and B. subtilis enzymes normally have Asp at position 87 (or 69), the B. subtilis ASL has Ile and Asp at 62 and 65, respectively, whereas human ASL has Glu and Arg at the equivalent positions. We have constructed, expressed, and purified the double mutant I62E/D65R as a "humanized" normal B. subtilis enzyme to compare with enzymes with a single mutation at position 62 (I62D/D65R), at position 69 (I62E/D65R/D69E), or at both positions (I62D/D65R/D69E). V(max) for conversion of adenylosuccinate to AMP and fumarate is 0.57 micromol/min/mg for I62E/D65R, 0.064 micromol/min/mg for I62D/D65R, 0.27 micromol/min/mg for I62E/D65R/D69E, and 0.069 micromol/min/mg for I62D/D65R/D69E. The K(m) for adenylosuccinate is elevated in the X62D mutants, and I62D/D65R is the least stable of these ASLs at 37 degrees C. The CD spectra of mutant and wild type enzymes are similar; thus, there are no appreciable structural changes. Clearly the Asp(62) causes the most drastic effect on ASL function, whereas the Glu(69) mutation produces only modest change. These results emphasize the importance of expanding tests for ASL deficiency to individuals with developmental delay of any severity, including individuals with autistic spectrum disorder. This study further demonstrates the usefulness of the B. subtilis ASL as a model to mimic the defective enzyme in ASL deficiency.