Sarafoglou, Kyriakie

Abstract Background Children with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency typically have height, weight, and body mass index (BMI) growth patterns that differ from the general population due to increased androgen and/or glucocorticoid exposures. With the recent surge in the development of new therapies, CAH-specific growth charts are needed to evaluate the effectiveness of these new treatments. Methods Retrospective data from patients aged 0 to 20 years with classic CAH, confirmed by hormonal testing and/or CYP21A2 genotyping, from 2 large clinical databases were analyzed. Specialized charts were developed using the lamda-mu-sigma semiparametric modeling method to generate CAH-specific percentile curves from 0 to 20 years. Nodal-point analyses were conducted to assess differences in incremental growth at 4, 8, 12, 16, and 20 years of age relative to Centers for Disease Control and Prevention (CDC) 2000 normative charts using 1-sided quantile tests and age of adiposity rebound estimated with curve derivative solutions. Results A total sample of 8692 visits from 515 patients was used. Growth (height-, weight-, BMI-for-age) channels of CAH patients were significantly different over the entire growing period and characterized by diminished pubertal spurt relative to the CDC reference. Onset of adiposity rebound based on BMI-for-age occurred earlier for CAH patients (females 3.3 years, males 3.9 years) compared to their normative counterparts (5-8 years). Conclusion Our study showed that at incremental time points throughout childhood, children with CAH collectively follow specific differences in growth trajectories as compared to unaffected children. These variations highlight the need for CAH-specific charts to assist in clinical management, appraisal of growth trajectories, and assessment of the impact of new therapies.

Abstract Context Single-tier newborn screening (NBS) for congenital adrenal hyperplasia (CAH) using 17-hydroxyprogesterone (17OHP) measured by fluoroimmunoassay (FIA) in samples collected at 24 to 48 hours produces a high false-positive rate (FPR). Second-tier steroid testing can reduce the FPR and has been widely implemented. Objective We investigated the accuracy of an alternative multitier CAH NBS protocol that incorporates molecular testing of the CYP21A2 gene and reduces the first-tier 17OHP cutoff to minimize missed cases. Methods We create a Minnesota-specific CYP21A2 pathogenic variants panel; developed a rapid, high-throughput multiplex, allele-specific-primer-extension assay; and performed a 1-year retrospective analysis of Minnesota NBS results comparing metrics between a conventional steroid-based 2-tier protocol and a molecular-based multitier NBS protocol, applied post hoc. Results CYP21A2 gene sequencing of 103 Minnesota families resulted in a Minnesota-specific panel of 21 pathogenic variants. The Centers for Disease Control and Prevention created a molecular assay with 100% accuracy and reproducibility. Two-tier steroid-based screening of 68 659 live births during 2015 resulted in 2 false negatives (FNs), 91 FPs, and 1 true positive (TP). A 3-tier protocol with a lower first-tier steroid cutoff, second-tier 21-variant CYP21A2 panel, and third-tier CYP21A2 sequencing would have resulted in 0 FNs, 52 FPs, and 3 TPs. Conclusion Incorporation of molecular testing could improve the accuracy of CAH NBS, although some distinct challenges of molecular testing may need to be considered before implementation by NBS programs.

BACKGROUND Diagnosis of PMM2-CDG, the most common congenital disorder of glycosylation (CDG), relies on measuring carbohydrate-deficient transferrin (CDT) and genetic testing. CDT tests have false negatives and may normalize with age. Site-specific changes in protein N-glycosylation have not been reported in sera in PMM2-CDG. METHODS Using multistep mass spectrometry–based N-glycoproteomics, we analyzed sera from 72 individuals to discover and validate glycopeptide alterations. We performed comprehensive tandem mass tag–based discovery experiments in well-characterized patients and controls. Next, we developed a method for rapid profiling of additional samples. Finally, targeted mass spectrometry was used for validation in an independent set of samples in a blinded fashion. RESULTS Of the 3,342 N-glycopeptides identified, patients exhibited decrease in complex-type N-glycans and increase in truncated, mannose-rich, and hybrid species. We identified a glycopeptide from complement C4 carrying the glycan Man 5 GlcNAc 2 , which was not detected in controls, in 5 patients with normal CDT results, including 1 after liver transplant and 2 with a known genetic variant associated with mild disease, indicating greater sensitivity than CDT. It was detected by targeted analysis in 2 individuals with variants of uncertain significance in PMM2 . CONCLUSION Complement C4–derived Man 5 GlcNAc 2 glycopeptide could be a biomarker for accurate diagnosis and therapeutic monitoring of patients with PMM2-CDG and other CDGs. FUNDING U54NS115198 (Frontiers in Congenital Disorders of Glycosylation: NINDS; NCATS; Eunice Kennedy Shriver NICHD; Rare Disorders Consortium Disease Network); K08NS118119 (NINDS); Minnesota Partnership for Biotechnology and Medical Genomics; Rocket Fund; R01DK099551 (NIDDK); Mayo Clinic DERIVE Office; Mayo Clinic Center for Biomedical Discovery; IA/CRC/20/1/600002 (Center for Rare Disease Diagnosis, Research and Training; DBT/Wellcome Trust India Alliance)

AbstractBackgroundPhosphomannomutase 2 deficiency (PMM2‐CDG) affects glycosylation pathways such as the N‐glycosylation pathway, resulting in loss of function of multiple proteins. This disorder causes multisystem involvement with a high variability among patients. PMM2‐CDG is an autosomal recessive disorder, which can be caused by inheriting two pathogenic variants, de novo mutations or uniparental disomy.Case PresentationOur patient presented with multisystem symptoms at an early age including developmental delay, ataxia, and seizures. No diagnosis was obtained till the age of 31 years, when genetic testing was reinitiated. The patient was diagnosed with a complete maternal mixed hetero/isodisomy of chromosome 16, with a homozygous pathogenic PMM2 variant (p.Phe119Leu) causing PMM2‐CDG.A literature review revealed eight cases of uniparental disomy as an underlying cause of CDG, four of which are PMM2‐CDG.ConclusionSince the incidence of homozygosity for PMM2 variants is rare, we suggest further investigations for every homozygous PMM2‐CDG patient where the segregation does not fit. These investigations include testing for UPD or a deletion in one of the two alleles, as this will have an impact on recurrence risk in genetic counseling.

Glycine encephalopathy (GE), or nonketotic hyperglycinemia (NKH), is a rare recessive genetic disease caused by defective glycine cleavage and characterized by increased accumulation of glycine in all tissues. Here, based on new case reports of GLDC loss-of-function mutations in GE patients, we aimed to generate a zebrafish model of severe GE in order to unravel the molecular mechanism of the disease. Using CRISPR/Cas9, we knocked out the gldc gene and showed that gldc –/– fish recapitulate GE on a molecular level and present a motor phenotype reminiscent of severe GE symptoms. The molecular characterization of gldc –/– mutants showed a broad metabolic disturbance affecting amino acids and neurotransmitters other than glycine, with lactic acidosis at stages preceding death. Although a transient imbalance was found in cell proliferation in the brain of gldc –/– zebrafish, the main brain networks were not affected, thus suggesting that GE pathogenicity is mainly due to metabolic defects. We confirmed that the gldc –/– hypotonic phenotype is due to NMDA and glycine receptor overactivation, and demonstrated that gldc –/– larvae depict exacerbated hyperglycinemia at these synapses. Remarkably, we were able to rescue the motor dysfunction of gldc –/– larvae by counterbalancing pharmacologically or genetically the level of glycine at the synapse.

Summary Objectives To evaluate obesity and overweight in children with congenital adrenal hyperplasia ( CAH ) and associations with glucocorticoids, fludrocortisone and disease control. Adjusting body mass index‐for‐height‐age ( BMI HA ) percentile is proposed to correct misclassification of obese/overweight status in CAH children with advanced bone age and tall‐for‐age stature. Design Longitudinal. Patients One hundred and ninety‐four children with CAH seen from 1970 to 2013: 124 salt wasting ( SW ); 70 simple virilizing ( SV ); 102 females. Measurements Body mass index (BMI) end‐points were overweight (85–94 percentile) and obese (≥95 percentile). Results Approximately 50% of the children had at least one BMI measurement ≥95 percentile and about 70% had at least one ≥85 percentile. Using BMI HA percentiles, obesity incidence decreased slightly in SW children (47–43%) and markedly in SV children (50–33%); however, overweight status was not reduced. Only 6% of SW and 1% of SV children were persistently obese (≥3 clinic visits) when BMI HA was applied, whereas overweight status persisted in 35% of SW and 33% of SV children. Most obesity or overweight when using BMI HA occurred before age 10 and there was no association with hydrocortisone (HC) or fludrocortisone dosing. Adiposity rebound for SW children occurred by 3·3 years and in SV females by age 3·8 years, over a year earlier than the adiposity rebound for healthy children. Conclusion Children with CAH are at higher risk for early onset obesity and overweight with or without using BMI HA but rates of persistent obesity were lower than previously reported. Careful HC dosing during early childhood is needed to prevent increased weight gain and an early adiposity rebound.

Summary Objectives Estimates of high blood pressure ( BP ) incidence in children with congenital adrenal hyperplasia ( CAH ) vary widely; risk factors are poorly understood. We estimated incidence of hypertension by CAH subtype and sex, and assessed its association with body mass index, hydrocortisone and fludrocortisone. Design Longitudinal. Patients Chart review of 180 paediatric CAH patients (120 salt wasting; 60 simple virilizing; 93 females) seen from 1970 to 2013. Measurements High BP was diagnosed by diastolic or systolic blood pressure measurement ≥95th percentile for age, sex and height; hypertension was diagnosed with high BP on at least three clinic visits. Results Children with classic CAH who received fludrocortisone had a significantly higher rate of hypertension (55% vs 31%) than those who did not. Hypertension incidence was higher in salt‐wasting CAH (58%) than in simple‐virilizing CAH (35%). Hypertension first occurred before age 5 years in 91% of salt‐wasting males and 50% of cases in salt‐wasting females; most simple‐virilizing cases occurred during ages 10–18 years. Rates of hypertension were higher in children who had three or more measurements with 17‐ OHP < 400 ng/dl (12·12 nmol/l), and this difference was significant in salt‐wasting males. Children on fludrocortisone who had three or more readings of 17‐ OHP < 400 ng/dl (12·12 nmol/l) had a significantly higher rate of hypertension than those who did not. Hydrocortisone dose was not associated with hypertension. Conclusion Children with CAH are at higher risk for hypertension than the general paediatric population, and incidence differs by sex and CAH subtype. Hypertension was higher in children on fludrocortisone and who were oversuppressed.

Screened for by all state newborn screening (NBS) programs in the United States, mitochondrial acetoacetyl-coenzyme A thiolase (T2), or β-ketothiolase, deficiency is a rare autosomal recessive disorder that causes ketoacidosis and hypoglycemia/hyperglycemia. Outcomes vary from normal development to severe cognitive impairment or even death after an acute episode of ketoacidosis. The classical biochemical profile of T2 deficiency is a result of null mutations in both alleles of the ACAT1 gene and consists of persistently increased urinary excretion of ketones, characteristic organic acids, and tiglylglycine as well as abnormal blood or plasma acylcarnitine profiles in acute and stable conditions. Early diagnosis and aggressive management can prevent further episodes of ketoacidosis and lead to normal development. We report the cases of 3 children, all subsequently found to have mutations predicted to be associated with no residual T2 enzymatic activity, but only 1 was identified by NBS in Minnesota since 2001. To our knowledge, this is the first description of compound heterozygotes for null mutations associated with no enzymatic activity exhibiting normal urinary organic acid, blood, and plasma acylcarnitine profiles when clinically well, thereby explaining the false-negative NBS results. We suggest that T2 deficiency may be underrecognized, because the incidence of T2 deficiency in Minnesota, on the basis of these 3 cases, is 1 in 232 000, higher than the reported &lt;1 in 1 million incidence. Our cases emphasize that T2 deficiency must be considered in patients who present with ketoacidosis disproportionately severe to the triggering illness despite normal NBS results or nonspecific biochemical findings in blood and urine during asymptomatic periods.

SummaryNewborns identified with profound biotinidase deficiency (BTD) by the Minnesota Newborn Screening Program (MN NBS) between 1 October 2004 and 30 May 2008 were all from new immigrant groups. Thirty‐three positive cases of BTD were identified out of 264 727 infants screened by the Wolf colorimetric system during the period of this study by MN NBS. Five cases of profound BTD (0.1 to <0.6 nmol/min per ml) and 26 cases of partial BTD (0.9 to 2.3 nmol/min per ml) were later confirmed through measurement of serum biotinidase activity. The incidence of combined partial and profound BTD of 1/8540 and that of profound BTD of 1/52 945 in Minnesota are unusually high in comparison with the reported worldwide numbers of 1/61 067 for combined BTD and 1/137 401 for profound BTD. Four out of the 5 cases of profound BTD ascertained in the MN NBS cohort were of Somali ethnic background, and the remaining case was of Asian (Pakistani/Indian) ethnic background. All four Somali patients have the P497S mutation, with one of the four being homozygous for the mutation. The three compound heterozygotes all have a novel mutation (P142T) and two of them have another change (Y428Y) that has never been described. Within the last two decades, Minnesota has become home to an estimated 40 000 Somali immigrants and their children (<1% of the total Minnesota population). New population demographics prompt careful analysis of case cohorts to identify specific groups at risk for rare inborn errors of metabolism.