Analysis of variants in GATA4 and FOG2/ZFPM2 demonstrates benign contribution to 46,XY disorders of sex development.

BACKGROUND: GATA-binding protein 4 (GATA4) and Friend of GATA 2 protein (FOG2, also known as ZFPM2) form a heterodimer complex that has been shown to influence transcription of genes in a number of developmental systems. Recent evidence has also shown these genes play a role in gonadal sexual differentiation in humans. Previously we identified four variants in GATA4 and an unexpectedly large number of variants in ZFPM2 in a cohort of individuals with 46,XY Differences/Disorders of Sex Development (DSD) (Eggers et al, Genome Biology, 2016; 17: 243). METHOD: Here, we review variant curation and test the functional activity of GATA4 and ZFPM2 variants. We assess variant transcriptional activity on gonadal specific promoters (Sox9 and AMH) and variant protein-protein interactions. RESULTS: Our findings support that the majority of GATA4 and ZFPM2 variants we identified are benign in their contribution to 46,XY DSD. Indeed, only one variant, in the conserved N-terminal zinc finger of GATA4, was considered pathogenic, with functional analysis confirming differences in its ability to regulate Sox9 and AMH and in protein interaction with ZFPM2. CONCLUSIONS: Our study helps define the genetic factors contributing to 46,XY DSD and suggests that the majority of variants we identified in GATA4 and ZFPM2/FOG2 are not causative.

Functional analysis of novel desert hedgehog gene variants improves the clinical interpretation of genomic data and provides a more accurate diagnosis for patients with 46,XY differences of sex development.

BACKGROUND: Desert hedgehog (DHH) gene variants are known to cause 46,XY differences/disorders of sex development (DSD). We have identified six patients with 46,XY DSD with seven novel DHH gene variants. Many of these variants were classified as variants of uncertain significance due to their heterozygosity or associated milder phenotype. To assess variant pathogenicity and to refine the spectrum of DSDs associated with this gene, we have carried out the first reported functional testing of DHH gene variant activity. METHODS: A cell co-culture method was used to assess DHH variant induction of Hedgehog signalling in cultured Leydig cells. Protein expression and subcellular localisation were also assessed for DHH variants using western blot and immunofluorescence. RESULTS: Our co-culture method provided a robust read-out of DHH gene variant activity, which correlated closely with patient phenotype severity. While biallelic DHH variants from patients with gonadal dysgenesis showed significant loss of activity, variants found as heterozygous in patients with milder phenotypes had no loss of activity when tested with a wild type allele. Taking these functional results into account improved clinical interpretation. CONCLUSION: Our findings suggest heterozygous DHH gene variants are unlikely to cause DSD, reaffirming that DHH is an autosomal recessive cause of 46,XY gonadal dysgenesis. Functional characterisation of novel DHH variants improves variant interpretation, leading to greater confidence in patient reporting and clinical management.

Precocious puberty in children.

OBJECTIVE: To determine the etiology of precocious puberty in children and to compare the clinical and laboratory parameters of central and peripheral precocious puberty. STUDY DESIGN: Cross-sectional study. PLACE AND DURATION OF STUDY: Endocrine Clinic at National Institute of Child Health, Karachi, from January 2009 to December 2011. METHODOLOGY: Children presenting with precocious puberty were included. The age of onset of puberty was documented. Clinical evaluation, Tanner staging, height, height SDS, weight, weight SDS, body mass index, bone age, pelvic USG, plasma estradiol level and GnRH stimulation were done. Ultrasound of adrenal glands, serum level of 17 hydroxyprogesterone, ACTH, Renin, aldosterone and testosterone were performed in children with peripheral precocious puberty. MRI of adrenal glands and gonads was done in patients with suspected tumor of that organ and MRI of brain was done in patients with central precocious puberty. Skeletal survey was done in patients with Mc Cune-Albright syndrome. RESULTS: CAH (81.8%) indentified as a main cause in peripheral percocious puberty and idiopathic (67.74%) in central precocious puberty. Eighty five patients were registered during this period. The conditions causing precocious puberty were central precocious puberty (36.47%), peripheral precocious puberty (38.82%), premature pubarche (10.58%) and premature thelarche (14.11%). There was a difference in the age of onset of puberty in case of central precocious puberty (mean=3, 2-6 years) versus peripheral precocious puberty (mean=5.25; 3.62 - 7.0 years). Children with central precocious puberty showed higher height SDS, weight SDS, FSH, LH than those with peripheral precocious puberty. CONCLUSION: Etiology in majority of cases with peripheral precocious puberty was congenital adrenal hyperplasia and idiopathic in central precocious puberty. Central precocious puberty children showed higher height SDS, weight SDS, FSH, LH than peripheral precocious puberty.

Etiological diagnosis of undervirilized male/XY disorder of sex development.

OBJECTIVE: To do clinical, hormonal and chromosomal analysis in undervirilized male / XY disorder of sex development and to make presumptive etiological diagnosis according to the new Disorder of Sex Development (DSD) classification system. STUDY DESIGN: Case series. PLACE AND DURATION OF STUDY: Endocrine Unit at National Institute of Child Health, Karachi, Pakistan, from January 2007 to December 2012. METHODOLOGY: Patients of suspected XY DSD / undervirilized male visiting endocrine clinic were enrolled in the study. Criteria suggested XY DSD include overt genital ambiguity, apparent female/male genitalia with inguinal/labial mass, apparent male genitalia with unilateral or bilateral non-palpable testes, micropenis and isolated hypospadias or with undescended testis. The older children who had delayed puberty were also evaluated with respect to DSD. As a part of evaluation of XY DSD, abdominopelvic ultrasound, karyotype, hormone measurement (testosterone, FSH, LH), FISH analysis with SRY probing, genitogram, laparoscopy, gonadal biopsy and HCG stimulation test were performed. Frequencies and percentages applied on categorical data whereas mean, median, standard deviation were calculated for continuous data. RESULTS: A total of 187 patients met the criteria of XY DSD. Age ranged from 1 month to 15 years, 55 (29.4%) presented in infancy, 104 (55.6%) between 1 and 10 years and 28 (15%) older than 10 years. Twenty five (13.4%) were raised as female and 162 as (86.6%) male. The main complaints were ambiguous genitalia, unilateral cryptorchidism, bilateral cryptorchidism, micropenis, delayed puberty, hypospadias, female like genitalia with gonads, inguinal mass. The karyotype was 46 XY in 183 (97.9%), 46 XX in 2 (1.1%), 47 XXY in 1 (0.5%), 45 X/46 XY in 1 (0.5%) patient. HCG stimulation test showed low testosterone response in 43 (23 %), high testosterone response in 62 (33.2%), partial testosterone response in 32 (17.1%) and normal testosterone response in 50 (26.7%). Genitogram was carried out in 86 (45.98%) patients. Presumptive etiological diagnosis of androgen sensitivity syndrome/ 5-alpha reductase deficiency, testicular biosynthetic defect/ leydig cell hypoplasia, partial gonadal dysgenesis, ovotesticular DSD, XX testicular DSD, mixed gonadal dysgenesis, testicular vanishing syndrome, klinefelter syndrome, hypogonadotropic hypogonadism, isolated hypospadias and isolated micropenis was made. CONCLUSION: Clinical, chromosomal and hormonal assessment may help in making the presumptive etiological diagnosis. Further molecular genetics analysis are needed in differentiating these abnormalities and to make a final diagnosis.

Effect of intravenous pamidronate treatment in children with osteogenesis imperfecta.

OBJECTIVE: To assess the beneficial effect of intravenous pamidronate treatment in children with osteogenesis imperfecta (OI). STUDY DESIGN: Experimental study. PLACE AND DURATION OF STUDY: Endocrine Unit at the National Institute of Child Health, Karachi, Pakistan, from January 2007 to December 2011. METHODOLOGY: All children diagnosed with osteogenesis imperfecta on the basis of repeated spontaneous fractures and typical radiological findings registered during the study period, were included in this study. Pamidronate therapy were offered to those with more than 3 fractures per year or had platyspondyly. Pamidronate disodium was diluted in isotonic saline and administered by slow ravenous infusion over 3 hours in a dosage 1 mg/kg/day for 3 consecutive days 3 monthly for 2 years. Fracture rate, bone mineral density (BMD), mobility score, wellbeing and pain episodes were evaluated at baseline and 2 years after the treatment. Good response was defined as less than 2 fractures per year or mobility score improvement and poor response as more than 2 fracture per year with mobility score less than 2. RESULTS: Seventy two patients were included in this study. There were 40 boys and 32 girls with mean age of 3.64 ± 3.2 years. The annual fracture rate decreased overall from 5.8 ± 1.61 to 0.6 ± 0.93 (p < 0.001). BMD Z-score improved from -5.3 ± 1.74 to -1.7 ± 0.72 (p < 0.001). Mobility score was 0.94 ± 1.30 at baseline and 2.5 ± 1.02 at the end of the treatment (p < 0.001). Wellbeing gained from 3.63 ± 1.44 to 7.8 ± 1.18 (p < 0.001) and pain episode improved from 24.1 ± 8.15 to 2.7 ± 8.31 (p < 0.001). Good response was noted in 92% of patients and poor response in 8% patients. CONCLUSION: Bisphosphonate seems to be an effective symptomatic treatment for children with osteogenesis imperfecta irrespective of severity of mutation or clinical phenotype. Cyclical bisphosphonate therapy has a positive effect on fracture rate, BMD, mobility score, wellbeing and pain episode.

Exome sequencing identifies CTSK mutations in patients originally diagnosed as intermediate osteopetrosis.

Autosomal Recessive Osteopetrosis is a genetic disorder characterized by increased bone density due to lack of resorption by the osteoclasts. Genetic studies have widely unraveled the molecular basis of the most severe forms, while cases of intermediate severity are more difficult to characterize, probably because of a large heterogeneity. Here, we describe the use of exome sequencing in the molecular diagnosis of 2 siblings initially thought to be affected by "intermediate osteopetrosis", which identified a homozygous mutation in the CTSK gene. Prompted by this finding, we tested by Sanger sequencing 25 additional patients addressed to us for recessive osteopetrosis and found CTSK mutations in 4 of them. In retrospect, their clinical and radiographic features were found to be compatible with, but not typical for, Pycnodysostosis. We sought to identify modifier genes that might have played a role in the clinical manifestation of the disease in these patients, but our results were not informative. In conclusion, we underline the difficulties of differential diagnosis in some patients whose clinical appearance does not fit the classical malignant or benign picture and recommend that CTSK gene be included in the molecular diagnosis of high bone density conditions.

Safety of insulin tolerance test for the assessment of growth hormone deficiency in children.

OBJECTIVE: To determine the safety of insulin tolerance test (ITT) for assessing growth hormone (GH) deficiency in children. METHODS: This hospital based study was conducted at the National Institute of Child Health, Karachi from 1st November 2008 till 30th October 2009. All children suspected of growth hormone deficiency, were included after excluding all other causes of short stature. Verbal informed consent was taken from the parents. Children less than 2 years of age, weighing less than 10 kg, untreated/inadequately treated hypothyroidism or Addison's disease, epilepsy, having history of hypoglycaemic fits or cardiac disease were excluded. All children were subjected to the international standard protocol of ITT and their samples of growth hormone and blood sugars were drawn. Complications during the procedure like hypoglycaemia, hypothermia, loss of consciousness, fits, vomiting and failure to achieve hypoglycaemia were recorded. Insulin tolerance test was performed on a total of 168 subjects. The data was entered in SPSS version 17 for analysis. RESULTS: A total of 168 children were subjected to the ITT. Four of them were abandoned as they could not achieve hypoglycaemia despite repeating the dose of insulin. Results were analyzed on 164 children whose mean age was 10 +/- 3.5 years, There were 96 (58%) males and 68 (41%) females. Over all 79.8% children achieved hypoglycaemia. None of the subjects developed any complication (fits, loss of consciousness,) or required intravenous glucose during the test and it was completed in all children with close monitoring. The results showed that there was a significant effect of time after insulin administration on both the blood glucose level (BG) and growth hormone (GH) levels. The blood glucose level decreased rapidly after administration of insulin and was lowest 30 minutes after injection and showed an increasing trend in subsequent readings, becoming almost equal to the baseline value 120 min after injection. From the study group 111 (66%) children were diagnosed as having growth hormone deficiency, 52 (31.3%) were normal and 1 (0.6%) had growth hormone insensitivity. CONCLUSION: ITT in children was found to be a safe and reliable test but can be potentially dangerous and requires very close monitoring and supervision and should be performed in a center with experienced staff.

Hyperinsulinemia and waist circumference in childhood metabolic syndrome.

OBJECTIVE: To determine the characteristics of obese children presenting at a tertiary care hospital and the frequency of metabolic syndrome (MS) in them using two paediatric definitions. STUDY DESIGN: Cross-sectional study. PLACE AND DURATION OF STUDY: The Endocrine Clinic of National Institute of Child Health, Karachi, from November 2005 till May 2008. METHODOLOGY: A total of 262 obese children aged 4-16 years, with BMI greater than 95th percentile were included. Children having obesity due to syndromes, medications causing weight gain, chronic illness and developmental disability were excluded. Blood pressure, waist circumference, fasting triglycerides, HDL, insulin and glucose levels were obtained. Obesity was defined as BMI > 95th percentile for age and gender according to the UK growth reference charts. The prevalence of metabolic syndrome was estimated using to the De Ferrantis and Lambert definitions. RESULTS: The frequency of MS varied between 16% and 52% depending on whether insulin levels were included in the definition. There was a significant positive correlation(r) when the metabolic parameters were correlated with waist circumference and insulin levels, except HDL which was negatively correlated. All the metabolic parameters like waist circumference, triglycerides, high density lipoprotein cholesterol and systolic blood pressure increased considerably across the insulin quartile (p < 0.05). The most noteworthy anthropometric and metabolic abnormality were the waist circumference (46.5%) and insulin levels (58%) respectively. CONCLUSION: There was a marked difference in the frequency of metabolic syndrome according to the definition used. The waist circumference and hyperinsulinemia are significant correlates of MS in obese children. There is a need for establishing normal insulin ranges according to age, gender and pubertal status. The clinical examination and investigations ought to include waist circumference and insulin levels together as a part of the definition of MS, for early detection and intervention of childhood obesity.

Effects of diabetes self-management education on glycaemic control in children with insulin-dependent diabetes mellitus.

OBJECTIVE: To evaluate the effect of diabetes self-management education (DSME) on glycaemic control (HbA1c) in Pakistani children suffering from type-1 diabetes mellitus. STUDY DESIGN: Quasi-experimental study. PLACE AND DURATION OF STUDY: This study was conducted at the Diabetic OPD of National Institute of Child Health, Karachi, from April to September 2009. METHODOLOGY: Sixty children with a mean age of 9.94 years with type-1 Diabetes mellitus (T1DM) were selected conveniently from the diabetic OPD. The patients along with their parents/caregivers attended a modular series of diabetes self-management education program consisting of 2 sessions. Customized program was designed to educate children regarding general information about the disease, basic insulin therapy, planning for hypoglycaemia, hyperglycaemia, activity, traveling and basic nutritional management. It was conducted by a multidisciplinary paediatric diabetes team including an endocrinologist, general paediatrician, nutritionist and diabetic nurse. The educational sessions were followed by monthly revision exercises. HbA1c levels were measured at baseline and after 3 months and compared using paired sample t-test. RESULTS: Out of a total of 60 patients, 50 completed the trial. There was a significant decrease in the HbA1c levels after the DSME program. The mean pre- and postintervention HbA1c levels were 9.67±0.65 and 8.49±0.53 respectively with a p-value < 0.001. CONCLUSION: In the studied group, DSME programs helped to improve glycaemic control. It should be an integral part of patient treatment in diabetic care setups.

Frequency, clinical characteristics and outcome of diabetic ketoacidosis in children with type-1 diabetes at a tertiary care hospital.

OBJECTIVE: To observe the frequency, demographic data and outcome of diabetic ketoacidosis (DKA) in children with established type 1 diabetes and newly diagnosed diabetes at a tertiary care hospital. METHODS: The case record review was done of children admitted with the diagnosis of DKA at The National Institute of Child Health, Karachi from 1st June 2008 till 31st May 2009. All records with the diagnosis of DKA were reviewed and those children with only hyperglycaemia, or who did not fulfill the criteria of DKA were excluded. The demographic data and laboratory investigations which included blood sugar monitoring, arterial blood gases, urine analysis especially for ketones, serum electrolytes, complete blood count and blood culture 'were reviewed. The previous numbers of admissions in children with established DKA were also noted with reasons. The duration of symptoms and fluids required, time of recovery, complications, and outcome were noted and compared between those with established diabetes and children with newly diagnosed diabetes. Data was entered and analyzed on SPSS version 15. RESULTS: Out of 124 case records, 117 were included which fulfilled the criteria of DKA. A large number, 65 (55.5%) children were in the > 10 years age group with a female predominance. Out of 117 children 50 (42.7%) had established Type 1 diabetes and 67 (57.2 %) children had newly diagnosed diabetes. The commonest presenting complaints in both groups were respiratory distress (87.1%) and vomiting (77.7%). The symptoms of polyuria, polydipsia and nocturia were more among the newly diagnosed children as compared to those with established diabetes with a significant p value <0.001. The comparison of clinical features and laboratory investigations of the two groups showed no difference except that those children with established diabetes improved earlier, required lesser duration of intravenous fluids and their insulin was changed to subcutaneous in less time compared with newly diagnosed children. (p < 0.001). The commonest complication in both groups was hypoglycaemia followed by hyponatraemia, more in newly diagnosed diabetic children. CONCLUSION: These soaring numbers are just from one center, highlighting the issue of this much neglected disease in our country. More studies on a larger scale are needed to assess the prevalence/incidence in our children and also more emphasis with educational programmes on prevention of recurrent attacks of DKA.