Angel O K Chan’s research while affiliated with Queen Mary Hospital and other places

Disorders of sex development (DSD) are defined as congenital conditions in which development of chromosomal, gonadal or anatomical sex is atypical. In 46, XY DSD, the genotype is XY, but the external genitalia is incompletely virilised, ambiguous, or completely female. The objectives of this prospective study are to evaluate the testicular Sertoli and Leydig cell functions, to establish the genetic basis and to determine the relative prevalence of etiologies in Chinese patients with 46,XY DSD in Hong Kong. All patients with 46,XY DSD (either new or known) presented to five paediatric departments in Hong Kong from July 2009 till June 2011 were recruited. They were assessed by paediatric endocrinologists. Comprehensive evaluation of testicular and adrenal functions was performed using serum hormonal assays and urine steroid profiling. Based on the hormonal results, mutational analyses of the candidate genes by polymerase chain reaction and direct DNA sequencing were conducted to delineate the genetic basis of the etiologies. Sixty-five patients (54 male and 11 female) with 46,XY DSD were recruited. Their age ranged from birth to 27 years. Sixty-one (94%) patients presented with ambiguous external genitalia, two presented with delayed puberty and one each with primary amenorrhoea and inguinal hernia. Definitive diagnoses were made in 25 (38%) patients. Eleven (17%) patients had 5-alpha reductase 2 deficiency. Androgen insensitivity was confirmed by genetic analysis in eight (12%) patients. There was one patient with each of the following etiologies: Swyer syndrome, SF-1 mutation, Frasier syndrome, cholesterol side-chain cleavage deficiency, persistent Mullerian duct syndrome and mixed gonadal dysgenesis. Genetic basis of the etiologies was delineated in 23 (35%) patients. A total of 10 novel mutations were identified. The longest follow up period was 27 years, none of the patients requested change of gender sex so far. In conclusion, 46,XY DSD is a heterogeneous group with diverse etiologies. Although 5-alpha reductase 2 deficiency is believed to be rare, it is not uncommon in Hong Kong.

Barakat syndrome, also known as HDR syndrome (hypoparathyroidism, deafness and renal dysplasia), is a rare autosomal dominant disorder, secondary to muta- tion of the GATA3 gene which is located at chromosome 10p. The GATA3 protein is one of the transcription factors which play an essential role in the embryonic development of the parathyroids, inner ears and kidneys. We report a Chinese patient who presented with hypocalcaemic convulsion at day 11 of life due to pri- mary hypoparathyroidism. Her serum calcium became normalized with calcium and vitamin D supplement. There was no other clinical feature to suggest DiGeorge syndrome and no family history of hypocalcaemia. The exact cause of hypoparathyroidism was not known at that time. At 6 months of age, she developed the first episode of E-coli urinary tract infection. Ultrasonogram of the kidney was normal but voiding cystogram revealed an intra-renal reflux of right kidney with dilated right ureter. Prophylactic antibiotics was pre- scribed. However, she developed repeated urinary tract infection with febrile seizures at 13 months and 15 months old respectively. Re-implantation of the right ureter was hence performed at 2 years of age. At the same time, she failed hearing screening test at her regu- lar health assessment and audiometry showed bilateral hearing deficit. When she was reassessed at ten years of age, she enjoyed good health with only mildly deranged renal function. In view of the presence of hypoparathyroidism, sensori- neural hearing deficit and renal anomaly, Barakat syn- drome was suspected and genetic analysis of the GATA3 gene was performed at nine years of age. A heterozygous novel deletion mutation (c.925-3_925-2delCA) was detected in intron 3 of the GATA3 gene. This mutation was not detected in her parents, suggesting that it is a de novo mutation. In conclusion, this is the first case report of a southern Chinese patient with Barakat syndrome diagnosed nine years after the initial presentation with hypoparathyroidism. Barakat syndrome is an extremely rare clinical entity and delayed diagnosis is not uncom- mon. Physicians should be aware of this condition for early diagnosis and family screening.

Background: 5α-Reductase 2 deficiency (5ARD) is a known cause of 46,XY disorders of sex development (DSD). Traditionally, the diagnosis relies on dihydrotestosterone (DHT) measurement, but the results are often equivocal, potentially leading to misdiagnosis. We reviewed alternative approaches for diagnosis of 5ARD. Methods: We conducted a retrospective review of the results of urinary steroid profiling (USP) by GC-MS and mutational analysis of SRD5A2 [steroid-5-alpha-reductase, alpha polypeptide 2 (3-oxo-5 alpha-steroid delta 4-dehydrogenase alpha 2)] by PCR and direct DNA sequencing of all 46,XY DSD patients referred to our laboratory with biochemical and/or genetic findings compatible with 5ARD. We also performed a literature review on the laboratory findings of all 5ARD cases reported in the past 10 years. Results: Of 16 patients diagnosed with 5ARD between January 2003 and July 2012, 15 underwent USP, and all showed characteristically low 5α- to 5β-reduced steroid metabolite ratios. Four patients had DHT measured, but 2 did not reach the diagnostic cutoff. In all 12 patients who underwent genetic analysis, 2 mutations of the SRD5A2 gene were detected to confirm the diagnosis. Twenty-four publications involving 149 patients with 5ARD were published in the review period. Fewer than half of these patients had DHT tested. Nearly 95% of them had the diagnosis confirmed genetically. Conclusions: 5ARD can be confidently diagnosed by USP at 3 months postnatally and confirmed by mutational analysis of SRD5A2. Interpretation of DHT results may be problematic and is not essential in the diagnosis of 5ARD. We propose new diagnostic algorithms for 46,XY DSD.

Background: Deficiency in any one of the steroidogenic enzymes may result in congenital adrenal hyperplasia (CAH) and disorders of sex development (DSD). Urinary steroid profiling (USP) can quantify metabolites of all relevant steroids simultaneously in a single analysis and has established clinical applications in the investigation and diagnosis in these disorders. Patients and methods: A retrospective review was performed on all the samples sent to the Chemical Pathology Laboratory, Queen Elizabeth Hospital, Hong Kong, for the investigation of suspected disorders in steroid metabolism by USP between 2003 and 2011. Results: 432 patients had urine samples sent to our laboratory for USP for the investigation of CAH and DSD in the review period. USP showed diagnostic pattern of 21-hydroxylase deficiency (n=21), 5α-reductase 2 deficiency (n=12), 17α-hydroxylase deficiency (n=3), isolated 17,20-lyase deficiency (n=1), 11β-hydroxylase deficiency (n=1) and P450 oxidoreductase deficiency (n=1). Conclusions: 21-hydroxylase deficiency is the most common form of CAH while 5α-reductase 2 deficiency is the most common cause of 46,XY DSD in our population. USP is a useful tool in the investigation and diagnosis of CAH and DSD due to different steroidogenesis defects and should be included as a first-line endocrine investigation in this group of patients.

Context Cytochrome P450 side-chain cleavage enzyme (CYP11A1) catalyses the first and rate-limiting step of steroidogenesis, the conversion of cholesterol to pregnenolone. CYP11A1 deficiency is commonly associated with adrenal insufficiency, and in 46,XY individuals, with variable degrees of disorder of sex development (DSD). Patient and methods The patient was born with hyperpigmentation, micropenis, penoscrotal hypospadias, and mild cryptorchidism. Biochemical and hormonal findings were normal except for low testosterone and low-borderline cortisol. However, no short synacthen test was undertaken. Development was unremarkable apart from an episode labeled as sepsis with documented hyperkalemia and elevated C-reactive protein at age 15 days. Diagnosis of 46,XY DSD was made at age 2.5 months. Progression of hyperpigmentation prompted further investigations and the diagnosis of adrenal insufficiency was established at 2 years with raised ACTH, normal renin activity, and failure of cortisol to respond to short synacthen test. Genetic analyses were performed. The novel CYP11A1 mutations were characterized in vitro and in silico. Results The patient was compound heterozygous for two novel CYP11A1 mutations, p.R360W and p.R405X. p.R360W retained 30–40% of wild-type activity. In silico analyses confirmed these findings and indicated that p.R405X is severe. Conclusions This study demonstrates the pathogenicity of two novel CYP11A1 mutations found in a patient with delayed diagnosis of CYP11A1 deficiency. Patients with partial deficiencies of steroidogenic enzymes are at risk to be misdiagnosed if adrenal function is not assessed. The adrenocortical function should be routinely assessed in all patients with DSD including severe hypospadias of unknown origin to prevent life-threatening adrenal crises.

To the Editor: The steroid metabolic disorder 21-hydroxylase deficiency (21OHD)1 is increasingly being included in blood spot–based newborn-screening programs. Screening most commonly relies on 17-hydroxyprogesterone immunoassay, but it gives a high rate of false positives and may not provide differentiation from 2 other types of congenital adrenal hyperplasia—P450 oxidoreductase deficiency (PORD) and 11-hydroxylase deficiency. Koyama et al. recently reported in Clinical Chemistry reference cutoffs for 4 urinary steroid metabolites and derived ratios for differentiating 21OHD and PORD (1). Although this report was based on samples from 46 institutions, they did not specify whether samples were obtained during second-line screening after a finding of an increased 17-hydroxyprogesterone result in a blood spot screen. It is useful that the authors provided genotypes for all patients positive for one of …

We report on an adult patient with citrin deficiency in Hong Kong, in whom a novel mutation was identified. The patient presented with recurrent hyperammonaemic encephalopathy due to impairment of the liver urea cycle enzyme argininosuccinate synthetase. This autosomal recessive condition is also characterised by interesting food preferences, notably aversion to carbohydrates and craving for protein-rich and/or lipid-rich foods, as well as neuropsychiatric symptoms. Plasma amino acid analysis is very useful in revealing urea cycle disorders, and mutational analysis of the SLC25A13 gene can confirm the diagnosis.

Congenital adrenal hyperplasia (CAH) caused by 21-hydroxylase deficiency (21OHD) is an autosomal recessive disorder due to mutation in the CYP21A2 gene. To elucidate the genetic basis of 21-hydroxylase-deficient CAH in Hong Kong Chinese patients. Mutational analysis of the CYP21A2 gene was performed on 35 Hong Kong Chinese patients with 21OHD using direct DNA sequencing and multiplex ligation-dependent probe amplification (MLPA). The genetic findings of 21 male and 14 female patients are the following: c.293-13A/C>G (intron 2 splice site; 20 alleles), p.I172N (13), p.R356W (7), p.Q318X (4). A total of 20 mutant alleles contained gross deletion/conversion of all or part of the CYP21A2 gene. A novel mutation, c.1367delA (p.D456fs), was detected in one patient. One patient had only a heterozygous mutation detected. Out of 35 patients, 16 would have been incorrectly genotyped if either DNA sequencing or MLPA alone was used for molecular analysis. The frequency of various mutations in the studied patients differs from those reported in other Asian populations. Gross deletion/conversion accounts for nearly one-third of the genetic defects. Therefore, laboratories must include methods for detecting point mutations as well as gross deletions/conversions to avoid misinterpretation of genotype. Genotyping has increasingly been proven to be a useful tool for supplementing, if not replacing, hormonal profiling for the diagnosis of 21OHD.