Pseudohypoparathyroidism (PHP) is characterised by hypocalcaemia and hyperphosphataemia due to resistance to parathyroid hormone (PTH). This was the first hormone resistance syndrome described in 1942 by Fuller Albright and his colleagues.1

There are three forms of PHP, namely: PHP-1, PHP-2, and pseudopseudohypoparathyroidism (PPHP). Evidently, PHP-1 differs from PHP-2 in that patients with the former show a blunted urinary cyclic AMP (cAMP) response to exogenous administration of PTH, whereas those with PHP-2 have normal urinary cAMP excretion but a blunted phosphaturic response. Moreover, PHP-1 is further classified into three different subtypes (1a, 1b, and 1c) based on the presence or absence of Albright’s hereditary osteodystrophy (AHO), which typically includes short stature, obesity, brachydactyly, ectopic ossification, and mental retardation. Both PHP-1a and PHP-1c display features of AHO, but PHP-1b does not. Furthermore, PHP-1a is distinguished from PHP-1c in that it contains the inactivating mutation in the gene encoding Gsα (GNAS).

Patients with both PHP-1a and PPHP carry heterozygous inactivating GNAS mutations. Apart from having AHO, patients with PHP-1a show resistance to hormones that act via G protein–coupled receptors. Patients with PPHP show only the features of AHO.

A 44-year-old woman, with no significant past medical illness, presented to the Department of Orthopaedics and Traumatology of Caritas Medical Centre in 2006 because of progressive weakness and numbness in both lower limbs. These symptoms had been present for years and she was only recently unable to walk. Magnetic resonance imaging of the cervical spine revealed osteophytosis and thickening of posterior longitudinal ligament, resulting in narrowing of the spinal canal at multiple levels with compression on the cervical cord. She was diagnosed as having cervical spondylosis, and laminoplasty was performed in September 2007. Postoperatively, she was noted to have hypocalcaemia with a total serum calcium level of 1.81 mmol/L (reference range [RR], 2.10-2.60 mmol/L), and a serum phosphate level of 1.08 mmol/L (RR, 0.8-1.5 mmol/L). The serum PTH level was 258 pg/mL (RR, 11-54 pg/mL); her adjusted calcium level was 2.12 mmol/L.

The patient was brought up by her stepmother since she was young. She got divorced and had no siblings and lost contact with her biological parents and their families. She also had a history of oligomenorrhoea since menarche with only one to two menstrual periods per year. On physical examination, her body weight was 87.2 kg and height 1.61 m, with a body mass index of 33.6 kg/m². She was obese with a moon face, but there was no definite brachydactyly. The clinical diagnosis was PHP.

Other investigations revealed that she had subclinical hypothyroidism with a serum thyroid-stimulating hormone (TSH) level of 7.64 mIU/L (RR, 0.50-4.70 mIU/L) and serum free T₄ level of 10.7 pmol/L (RR, 9.1-23.8 pmol/L). The anti-thyroglobulin antibody titre was < 1/100 but the anti-microsomal antibody titre was 1/24 600. Her serum follicle-stimulating hormone level was 20 U/L, serum luteinising hormone level was 14.8 U/L, and serum oestradiol level was <73 pmol/L. The short synacthen test (using 250 µg tetracosactrin) showed a baseline serum cortisol level of 77 nmol/L and peak level of 500 nmol/L. She was started on calcitriol and calcium supplement as well as thyroxine replacement.

She only had one child, a 16-year-old son who also had “calcium problem”. He was followed up by the Department of Paediatrics and Adolescent Medicine of our hospital. In 2003, he had presented with a generalised tonic-clonic convulsion at the age of 12 years. At that time, his serum calcium level was 1.46 mmol/L, phosphate level of 1.98 mmol/L, and a PTH level of 70 pg/mL. The thyroid function test was normal. He was obese and tall with a body weight of 60 kg (at 97th percentile in the growth chart) and height of 166.3 cm (>97th percentile). He suffered from mild mental retardation and studied in special school. There was mild shortening of the fourth and fifth metacarpals (Fig). He was diagnosed as having PHP with AHO features, and received calcitriol and calcium supplement. Over the years, he had gone through a normal puberty. The brachydactyly had become more prominent.

We performed a mutation analysis on the GNAS gene of both the mother and her son. Genomic DNA of both patients was extracted from peripheral blood leukocytes using the QIAamp Blood Kit (Qiagen, Hilden, Germany). The coding exons and the flanking regions of the SPG4 gene were amplified using a polymerase chain reaction and sequenced. The numbering of nucleotides was based on GenBank accession number NM_000516.4 with 394 amino acids. Protocol is available on request.

A heterozygous missense mutation, NM_000516.4(GNAS):c.682C>T (p.Arg228Cys), in the GNAS gene was identified in both the proband and her son. This was a novel mutation, with involvement of a structurally non-conservative substitution of the evolutionary conserved amino acid change predicted to affect protein function by Sorting Intolerant From Tolerant analysis, Polyphen-2 and MutationTaster analyses. Screening in 300 normal chromosomes did not suggest this as a polymorphic site. The diagnosis of the mother was PHP-1a with mild AHO, and of her son was PHP-1a with AHO.

It appears that PHP-1a is an autosomal dominant disease in which full clinical and metabolic abnormalities may not be present initially, but become apparent later. Patients with PHP-1a showed a heterozygous inactivating germline mutation in GNAS, the gene encoding the α-subunit of the stimulatory GTP binding protein (Gsα). This could lead to a reduced Gsα protein level and cellular activity and thus the clinical resistance phenotype.2

The GNAS gene maps to 20q13 and contains 13 exons.3 The mutation can be localised in the entire coding region of the gene. All exons can be affected by loss-of-function alterations with the exception of exon 3, where no mutations have been detected to date.4 The hot-spot mutations accounting for about 20% of all mutations so far described have been identified on exon 7.5 As for the types of mutations, small insertions, deletions and amino-acid substitutions predominate. Our patient showed a novel heterozygous missense mutation of exon 9 in the GNAS gene, which is considered to be functionally deleterious.

Maternal inheritance of this GNAS mutation leads to PHP-1a (ie AHO plus hormone resistance), while paternal inheritance of the same mutation leads to PPHP (ie AHO only).6 This imprinted mode of inheritance for hormone resistance can be explained by the predominantly maternal expression of Gsα in certain tissues, including renal proximal tubules.7 In our case, the son inherited the same molecular defect from his mother, resulting in PHP-1a. It could be postulated that the proband should also inherit the condition from her mother, but this remains to be substantiated as there is not much helpful information available.

Our patient (the mother) also had subclinical hypothyroidism; the anti-microsomal antibody was positive. She had evidence of hypogonadism (oligomenorrhoea and low oestradiol levels). By contrast, her son had normal thyroid function, and he had a normal puberty. Co-existing endocrinological abnormalities may ensue, as individuals with PHP-1a also demonstrate resistance to other hormones such as TSH, gonadotropins, and growth hormone-releasing hormone. Fernandez-Rebollo et al8 showed that most patients with PHP-1a have TSH resistance, which is usually mild, and manifests during childhood or adolescence. Goitre and anti-thyroid antibodies are usually absent.9 Clinical evidence of hypogonadism is common in PHP-1a, particularly in females, and manifests as delayed sexual maturation, amenorrhoea, oligomenorrhoea, and/or infertility. Affected individuals usually have slight hypo-estrogenism, but no definite evidence of increased basal or gonadotropin-releasing hormone–stimulated levels of circulating gonadotropins.10 Mantovani and Spada11 demonstrated that growth hormone deficiency is also common in patients with PHP-1a. However, the relevance of growth hormone deficiency on final height and obesity in these patients is not certain, because PPHP patients (who do not have hormonal resistance) also have short statures together with obesity. That study also evaluated the adrenocortical and corticotropin responsiveness in patients with PHP-1a; all of whom showed a normal response to 1 µg adrenocorticotropin and to corticotropin-releasing factor. Normal pituitary-adrenal function in these patients suggested that the presence of Gsα imprinting within the pituitary gland is cell-type specific.12

Spinal cord compression is a rare neurological complication of PHP or PPHP, which is due to ossification of the posterior longitudinal ligament that may compress the spinal cord.13 Presentations include spastic paraparesis, tetraparesis, and urinary incontinence.14 Many such patients endure long-term disability despite neurosurgical intervention. Our patient had a long history of neurological symptoms but presented late. After laminoplasty, she still had residual weakness. Hence, spinal cord compression should also be considered in patients with PHP or PPHP who present with neurological symptoms.

We have demonstrated a novel mutation in the GNAS gene in a small Chinese family with PHP-1a. One family member presented with spinal cord compression, which is a rare complication in PHP caused by ectopic ossification. Moreover, associated endocrinopathies, especially hypothyroidism and hypogonadism, are common in PHP-1a.

1. Albright F, Burnett CH, Smith PH, Parson W. Pseudohypoparathyroidism—an example of ‘Seabright-Bantam syndrome’. Endocrinology 1942;30:922-32.

2. Thakker RV. Genetic developments in hypoparathyroidism. Lancet 2001;357:974-6. CrossRef

3. Pattern JL, Johns DR, Valle D, et al. Mutation in the gene encoding the stimulatory G protein of adenylate cyclase in Albright’s hereditary osteodystrophy. N Engl J Med 1990;322:1412-9. CrossRef

4. Mantovani G, Spada A. Mutations in the Gs alpha gene causing hormone resistance. Best Pract Res Clin Endocrinol Metab 2006;20:501-13. CrossRef

5. Yu S, Yu D, Hainline BE, et al. A deletion hot-spot in exon 7 of the Gs alpha gene (GNAS1) in patients with Albright hereditary osteodystrophy. Hum Mol Genet 1995;4:2001-2. CrossRef

6. Bastepe M. The GNAS locus and pseudohypoparathyroidism. Adv Exp Med Biol 2008;626:27-40. CrossRef

7. Bastepe M, Jüppner H. Pseudohypoparathyroidism. New insights into an old disease. Endocrinol Metab Clin North Am 2000;29:569-89. CrossRef

8. Fernandez-Rebollo E, Barrio R, Pérez-Nanclares G, et al. New mutation type in pseudohypoparathyroidism type 1a. Clin Endocrinol (Oxf) 2008;69:705-12. CrossRef

9. Weinstein LS, Yu S, Warner DR, Liu J. Endocrine manifestations of stimulatory G protein alpha-subunit mutations and the role of genomic imprinting. Endocr Rev 2001;22:675-705.

10. Namnoum AB, Marriam GR, Moses AM, Levine MA. Reproductive dysfunction in women with Albright’s hereditary osteodystrophy. J Clin Endocrinol Metab 1998;83:824-9.

11. Mantovani G, Spada A. Resistance to growth hormone releasing hormone and gonadotropins in Albright’s hereditary osteodystrophy. J Paediatr Endocrinol Metab 2006;19:663-70. CrossRef

12. Mantovani G, Maghnie M, Weber G, et al. Growth hormone-releasing hormone resistance in pseudohypoparathyroidism type Ia: new evidence of imprinting for the Gs alpha gene. J Clin Endocrinol Metab 2003;88:4070-4. CrossRef

13. Iwase T, Nokura K, Mizuno T, Inagaki T. Spastic tetraparesis in a patient with pseudopseudohypoparathyroidism. J Neurol 2002;249:1457-8.

14. Alam SM, Kelly W. Spinal cord compression associated with pseudohypoparathyroidism. J R Soc Med 1990;83:50-1.

In this report, we present a couple who requested a double-factor pre-implantation genetic diagnosis (PGD) for both reciprocal translocation and alpha-thalassaemia.

Our patient, aged 36 years, enjoyed good past health and attended the subfertility clinic for recurrent miscarriage (5 times within 8 years). She had four spontaneous conceptions between 1997 and 2004 but all ended as first-trimester miscarriages. After 2004, she suffered from secondary subfertility and conceived again in 2007 following ovarian stimulation and intrauterine insemination. The fifth pregnancy again ended with first-trimester miscarriage.

She was subsequently referred to a clinical geneticist and found to be a carrier of a balanced reciprocal translocation 46,XX,t(2;10)(q33;q21.2). The husband had normal karyotypes and other relevant investigations for recurrent miscarriage were all negative. Both partners were alpha-thalassaemia trait carriers (South East Asia [SEA] type) as the genotype report revealed heterozygous alpha SEA type deletion. They were therefore referred to us for PGD.

Baseline investigations showed early follicular follicle-stimulating hormone levels of 6.5 IU/L and an antral follicle count of 19. The couple was counselled about the procedure and risks of PGD. It was decided to biopsy two blastomeres, so as to perform polymerase chain reaction (PCR) for alpha-thalassaemia on one of them, and carry out fluorescent in-situ hybridisation (FISH) for the reciprocal translocation on the other.

The first cycle of in-vitro fertilisation (IVF) and intra-cytoplasmic sperm injection (ICSI) was carried out in November 2010. After 10 days of ovarian stimulation, 12 oocytes were retrieved and 11 were in metaphase II for ICSI. Ten were normally fertilised and seven day-3 embryos were available for embryo biopsy. Two embryos were subsequently shown to be normal for FISH signals and either normal or heterozygous for alpha-thalassaemia SEA deletion. On day 5, there was only one fair-quality embryo at the morula stage for transfer, but the patient failed to conceive in that cycle.

She underwent a second IVF/ICSI/PGD cycle in January 2012. After 11 days of ovarian stimulation, 13 oocytes were retrieved. Twelve were fertilised, and eight day-3 embryos were available for embryo biopsy. One embryo was found to be balanced for the FISH signals and heterozygous for alpha-thalassaemia SEA deletion. That embryo developed to a good-quality blastocyst of grade 5BB and was transferred. She was pregnant but refused prenatal invasive testing in the second trimester because of the risk of miscarriage. She delivered a baby boy in October 2012 and the cord blood analysis confirmed the diagnosis of alpha-thalassaemia-1 carrier status with a normal karyotype 46,XY.

The Gap-PCR approach was used to amplify the alpha-SEA type deletion junction directly (Fig 1). Briefly, the normal allele was amplified by primers Zdel-1 and Zdel-2 (317 bp), but not by Zdel-1 and Xdel-3, because they were too far apart. In the alpha SEA deletion, the binding site for Zdel-2 was deleted, and that for Zdel-1 and Xdel-3 were brought into close proximity producing a PCR product of 280 bp. Linkage analysis was performed with fluorescent-labelled intragenic informative markers (16pTEL05 and 16pTEL06) and linked short tandem repeats (STR) markers within 2Mb flanking the alpha-globin loci (D16S521 and D16S3395). The relative positions of primers and markers around the alpha SEA deletion are shown in Figure 1. Single cell protocols, using multiple displacement amplification (MDA) or SurePlex DNA amplification, have been validated using single lymphocytes from the couple.

Two blastomeres were biopsied from each of the good-quality day-3 embryos. One blastomere underwent whole genome amplification (WGA) and PCR for PGD on the alpha-thalassaemia loci. The second blastomere was lysed for translocation detection by FISH.

In the first PGD cycle, WGA was performed by the MDA method according to the protocol previously published.1 In the second cycle, SurePlex DNA amplification (BlueGnome) was adopted for WGA. One µL of WGA product was used for PCR in a final volume of 25 µL containing 1X PCR buffer with MgSO₄, 0.2 mM dNTPs, and 1U FastStart Taq DNA polymerase (Roche). 0.5 µL of PCR product was separated by an ABI 3500 genetic analyser with a GeneScan 500ROX-size standard (Applied Biosystems) and analysed by GeneMapper (v4.1; Applied Biosystems).

The second blastomere underwent FISH with Vysis probes Tel 2p (green), CEP10 (aqua), and Tel 10q (orange). The signals were interpreted independently by two scientists.

In the first PGD cycle, Gap-PCR and intragenic informative markers 16pTEL05 and 16pTEL06 were used for PGD. All seven biopsied blastomeres resulted in a conclusive diagnosis. In the second cycle, the PGD protocol was modified in a few ways. Firstly, WGA was performed using the SurePlex DNA amplification system. Secondly, Gap-PCR primers Zdel-1 and Zdel-2 were omitted, since they were poorly amplified in SurePlex WGA DNA. Finally, two additional linked STR markers (D16S521 and D16S3395) were used to improve the diagnosis rate. Linkages of these additional markers with the SEA deletion locus were established with the leftover WGA DNA from embryos obtained in the first cycle. All embryos that underwent PGD showed conclusive results.

The leftover WGA DNA in the second cycle of PGD was used for array comparative genomic hybridisation (aCGH, 24Sure+, BlueGnome) for the detection of translocation. All samples showed conclusive result, which was consistent with those after FISH (Fig 2).

Our unit has offered PGD treatment for monogenetic diseases for more than 10 years, starting in 2000 for alpha-thalassaemia. The FISH technique was then developed for translocation carriers and pre-implantation genetic aneuploidy screening. In this case report, the couple described was the first to request PGD for both reciprocal translocation and alpha thalassaemia. The couple firstly attended our unit for PGD in 2009 and at that time the FISH technique was still routinely used for translocation. We decided to have two blastomeres biopsied, and undertook PCR on one (for alpha-thalassaemia) and FISH on the other (for reciprocal translocation). It is well-known that two-blastomere biopsy is more detrimental than one-blastomere biopsy on the implantation and pregnancy rate after embryo transfer.2 Since 2008, there was emerging evidence regarding the use of array CGH in both translocation carriers and preimplantation aneuploidy screening.3 4 5 Using aCGH, it could obtain information on all 24 chromosomes to detect aneuploidy, which is common in early human embryos, other than in translocated genetic material.4 Recourse to WGA in aCGH allowed us to use a single blastomere for both diagnoses as the amplified products could also be used for PCR. We switched to using WGA with SurePlex. However, before we acquired the technique of aCGH for PGD of translocation in 2012, the couple requested the second treatment cycle because of advancing maternal age. Therefore FISH was used again in the second PGD cycle for translocation, as in the first cycle.

Later, we used the leftover WGA DNA from the second PGD cycle for translocation and aneuploidy detection, using aCGH 2 weeks after the PGD treatment cycles. All embryos with abnormal FISH signals showed abnormal aCGH results (Fig 2a). The normal embryo showed a normal signal with no aneuploidy detected after aCGH, which was performed immediately after the delivery of the baby boy (Fig 2b). Karyotyping on cord blood of the baby confirmed our PGD and aCGH results.

So far, there have been three case reports from the same group of investigators on the use of double-factor PGD.6 7 8 All of them involved couples at risk for one genetic disease only (cystic fibrosis, Von Hippel-Lindau syndrome, Lynch syndrome), but aneuploidy screening was performed to improve the implantation and pregnancy rates in those of advanced maternal age. Our patient was at risk for two genetic diseases, namely alpha-thalassaemia and reciprocal translocation. In the aforementioned case reports too, two cells were removed for PGD (either one polar body and one blastomere, or two blastomeres). Although we also had two blastomeres biopsied in the treatment cycle of our couple, we have validated a protocol with which double-factor PGD can be performed with one-blastomere biopsy. With the use of aCGH, it becomes feasible and practicable to use one blastomere for both the monogenetic disease diagnosis and aCGH for either translocation carriers or aneuploidy screening in at-risk couples. The turnaround time of our protocol was approximately 2 days, rendering the fresh cycle day-5 blastocyst feasible for transfer.

We report the first live birth after double-factor PGD for alpha-thalassaemia and reciprocal translocation. We have also validated a protocol for double-factor PGD, in which WGA DNA obtained from a single blastomere can be used for PCR-based PGD and aCGH.

1. Chow JF, Yeung WS, Lau EY, et al. Singleton birth after preimplantation genetic diagnosis for Huntington disease using whole genome amplification. Fertil Steril 2009;92:828.e7-10.

2. De Vos A, Staessen C, De Rycke M, et al. Impact of cleavage-stage embryo biopsy in view of PGD on human blastocyst implantation: a prospective cohort of single embryo transfers. Hum Reprod 2009;24:2988-96. CrossRef

3. Wells D, Alfarawati S, Fragouli E. Use of comprehensive chromosomal screening for embryo assessment: microarrays and CGH. Mol Hum Reprod 2008;14:703-10. CrossRef

4. Fiorentino F, Spizzichino L, Bono S, et al. PGD for reciprocal and Robertsonian translocations using array comparative genomic hybridization. Hum Reprod 2011;26:1925-35. CrossRef

5. Forman EJ, Tao X, Ferry KM, Taylor D, Treff NR, Scott RT Jr. Single embryo transfer with comprehensive chromosome screening results in improved ongoing pregnancy rates and decreased miscarriage rates. Hum Reprod 2012;27:1217-22. CrossRef

6. Obradors A, Fernández E, Oliver-Bonet M, et al. Birth of a healthy boy after a double factor PGD in a couple carrying a genetic disease and at risk for aneuploidy: case report. Hum Reprod 2008;23:1949-56. CrossRef

7. Obradors A, Fernández E, Rius M, et al. Outcome of twin babies free of Von Hippel-Lindau disease after a double-factor preimplantation genetic diagnosis: monogenetic mutation analysis and comprehensive aneuploidy screening. Ferti Steril 2009;91:933.e1-7.

8. Daina G, Ramos L, Obradors A, et al. First successful double-factor PGD for Lynch syndrome: monogenic analysis and comprehensive aneuploidy screening. Clin Genet 2012;84:70-3. CrossRef

In this report, we describe a case of pyrrolizidine alkaloid–related Budd-Chiari syndrome in Hong Kong. A 10-month-old boy presented with ascites, right pleural effusion, and hepatomegaly after consumption of herbal drinks for 3 months. His clinical (including imaging) features were compatible with Budd-Chiari syndrome. Budd-Chiari syndrome is a rare disease entity in paediatric patients. In our case, extensive workup performed to look for the underlying cause of Budd-Chiari syndrome was unrevealing, except for toxic pyrrolizidine alkaloid exposure in his herbal drinks.

Influenza and pneumococcus co-infection can cause severe morbidity and mortality. Usually, this entails influenza A, while infection by influenza B is rarely serious. The literature describes influenza A epidemics leading to prolific loss of lives, notably the 1918 epidemic was blamed for the deaths of 40 to 50 million people. In this report, four patients were infected by influenza B during the influenza epidemic of 2011/12 in Hong Kong. All of them were previously healthy and had no chronic diseases; they were admitted to the hospital due to influenza-like symptoms. They rapidly deteriorated with multi-organ failure, and were subsequently diagnosed to be infected with influenza B and streptococci that gave rise to severe pneumonia. Three of them were infected with Streptococcus pneumoniae and one with Streptococcus pyogenes. All of them had leukopenia, septic shock, and acute kidney injury; two of whom died despite aggressive antibiotic treatment and organ support in the intensive care unit. According to the literature, this is the second case report of severe invasive pneumococcal pneumonia secondary to influenza B infection.

A 91-year-old woman diagnosed to have an inoperable cholangiocarcinoma had an uncovered metal stent inserted for palliative drainage. About 1.5 years later, tumour ingrowth into the metal stent caused cholangitis. Intraductal radiofrequency ablation was applied to create local coagulative tumour necrosis and the necrotic tissue was removed via a balloon catheter. A plastic stent was inserted to empirically treat any ensuing potential bile duct injury. The patient was discharged without complication with good palliative drainage. Intraductal radiofrequency ablation is a new technique for the treatment of metal stent occlusion due to tumour ingrowths. This is the first case report of this relatively safe and feasible new technique for the treatment of tumour ingrowth into a metal stent used as palliation for malignant biliary obstruction.