Chinese talisman is a religious handwriting or calligraphy which is believed to possess magical powers for expelling evils and avoiding misfortune. It is usually obtained from Daoism religious practitioners.1 Some people believe that consuming burnt Chinese talisman ashes dissolved in water is useful in curing diseases. Here we report a case of lead exposure after prolonged intake of ashes from burnt Chinese talismans.

The patient was a 41-year-old woman. She presented with progressive weakness of four limbs with signs of upper motor neuron disease (MND) since March 2012. Electromyogram findings were compatible with diffuse anterior horn cell disorder. Motor neuron disease was clinically diagnosed by the treating neurologist. Knowing that no curative option exists for MND, she started using Chinese talismans obtained from a religious practitioner in China (Fig). She was instructed to burn the Chinese talismans to ashes and ingest the ashes dissolved in water 3 times daily. She continued this practice for about 1 month, until she believed that it was not useful for her illness. She was then found to have elevated blood lead level (BLL) of 1.83 µmol/L or 38 µg/dL (reference level, <0.48 µmol/L or <10 µg/dL) during routine screening for treatable causes of neuropathy. Her blood mercury level was normal. Blood lead level rechecked 2 weeks later was 2.61 µmol/L (54 µg/dL). Other than the neurological symptoms, the patient had no other clinical features of lead poisoning such as elevated blood pressure, anaemia with basophilic stippling, or gastro-intestinal symptoms. She was subsequently referred to the poison centre for assessment of lead exposure.

Detailed enquiry did not point to any well-known source of lead exposure. She had been working as a cleansing worker in a food-processing factory until she became sick. She had never worked in any mining industry, metal refinery, glass factory, or battery factory. She did not have a history of gunshot wound, nor did she have exposure to lead paint or ceramic craft. Her husband had normal BLL. Abdominal X-ray did not reveal lead-containing foreign materials in the gastro-intestinal tract. She had used several health supplements including vitamin preparations and ginkgo biloba extract. The lead levels of these health supplements were found to be undetectable. Our suspicions were aroused when the patient reported that the Chinese talismans were supposed to be written with cinnabar (硃砂), a red mercuric sulfide containing ore. Substitution of cinnabar with another red mineral, minium (鉛丹, lead tetroxide), when used as Chinese medicine, has been reported.2 3 Analysis of the Chinese talisman by inductively coupled plasma mass spectrometry revealed a very high lead concentration of 17 342 µg/g, thus confirming the source of lead exposure.

Her BLL was 2.82 µmol/L (59 µg/dL) about 2 months after stopping the use of the Chinese talismans. Although her neurological presentation was not typical of lead neurotoxicity,4 there have been case reports of lead poisoning mimicking MND.5 6 Moreover, an animal study showed that anterior horn cells were sensitive to lead toxicity.7 Chelation therapy with succimer (dimercaptosuccinic acid [DMSA]) was started in view of these possibilities. A standard course of DMSA 10 mg/kg 3 times daily for 5 days, followed by 2 times daily for 14 days was given.8 Her BLL taken at 6 weeks after completion of DMSA course was 0.7 µmol/L (14 µg/dL). No improvement of limb weakness was observed in the patient 8 weeks after completion of DMSA course. Further courses of DMSA were judged unnecessary. The patient continued to have medical follow-up for MND.

This case illustrates a rare source of lead exposure related to the religious practice of consuming burnt Chinese talisman ashes dissolved in water to cure a disease. The list of common sources of lead exposure such as occupational, environmental and recreational ones, can be found in general medicine and toxicology textbooks.4 Uncommon and exotic sources reported in the literature usually involve traditional medicines, cosmetics, and ingestion of lead-containing foreign bodies (eg bullet, necklace, fishing sinker).9 10 11

The use of cinnabar has been described in Daoism alchemy and traditional Chinese medicine.12 13 Both lead tetroxide and cinnabar are red in colour with similar appearance, and substitution of cinnabar with lead tetroxide in Chinese medicine has been reported.3 The reason for the substitution is uncertain but it could be due to mixing up or related to the higher cost of cinnabar.2 The toxicity of lead tetroxide is known since ancient times in China. Lead poisoning related to the topical use of lead tetroxide in Chinese medicine for chronic ulcer has been reported.12

Before the era of molecular genetics, lead poisoning was believed to be one of the possible causes of MND.14 15 Nowadays, with the identification of different genes implicated in MND, it is believed that genetic causes account for a significant proportion of the cases.16 Neurological presentation of mild lead poisoning includes tiredness, headache, insomnia, memory loss, and lessened interest in leisure activities. In severe cases, coma, seizures, and peripheral neuropathy are possible.4 Lead-induced peripheral neuropathy is typically a pure motor disorder with features including footdrop and wristdrop.4 Severe form of lead-induced peripheral neuropathy has been reported in causing generalised weakness mimicking MND.5 6 Unlike MND, lead-induced peripheral neuropathy is associated with increased body burden of lead, a temporal sequence between lead exposure and progression of muscle weakness, clinical stabilisation or remission after removal from exposure, and systemic involvement with other features of lead poisoning such as anaemia and gastro-intestinal disturbance.17

Chelation therapy is usually not indicated in asymptomatic adults with BLL of <3.36 µmol/L (70 µg/dL).4 18 Nevertheless, there is no established action level in the presence of underlying MND. As lead-induced peripheral neuropathy is a possible reversible cause in this patient, chelation therapy was offered despite only a moderate increase in BLL. The lack of clinical improvement after cessation of exposure and normalisation of BLL made the diagnosis of lead-induced peripheral neuropathy unlikely in this patient.

Ingestion of burnt Chinese talisman is a possible source of lead exposure. This rare source of lead poisoning should be considered in a specific group of patients believing in this religious practice.

1. Wu YM. Talismans and spells. Available from: http://taiwanpedia.culture.tw/en/content?ID=2073. Accessed 18 Sep 2013.

2. Ban of cinnabar in Chinese medicine use in Taiwan [in Chinese]. Available from: http://www.twtcm.com.tw/law-content.php?id=9. Accessed 26 Jun 2014.

3. Lead and mercury content of proprietary Chinese medicine Babao powder [in Chinese]. Available from: http://www.consumers.org.tw/unit412.aspx?id=459. Accessed 11 Dec 2013.

4. Nelson LS, Lewin NA, Howland MA, Hoffman RS, Goldfrank LR, Flomenbaum NE. Goldfrank’s toxicological emergencies, 9th ed. New York, NY: McGraw-Hill Medical; 2011: 1266-83.

5. Boothby JA, DeJesus PV, Rowland LP. Reversible forms of motor neuron disease. Lead “neuritis”. Arch Neurol 1974;31:18-23. CrossRef

6. Rubens O, Logina I, Kravale I, Eglîte M, Donaghy M. Peripheral neuropathy in chronic occupational inorganic lead exposure: a clinical and electrophysiological study. J Neurol Neurosurg Psychiatry 2001;71:200-4. CrossRef

7. Yokoyama K, Araki S, Akabayashi A, Kato T, Sakai T, Sato H. Alternation of glucose metabolism in the spinal cord of experimental lead poisoning rats: microdetermination of glucose utilization rate and distribution volume. Ind Health 2000;38:221-3. CrossRef

8. Rogan WJ, Dietrich KN, Ware JH, et al. The effect of chelation therapy with succimer on neuropsychological development in children exposed to lead. N Engl J Med 2001;344:1421-6. CrossRef

9. Karri SK, Saper RB, Kales SN. Lead encephalopathy due to traditional medicines. Curr Drug Saf 2008;3:54-9. CrossRef

10. Levin R, Brown MJ, Kashtock ME, et al. Lead exposures in U.S. children, 2008: implications for prevention. Environ Health Perspect 2008;116:1285-93. CrossRef

11. St Clair WS, Benjamin J. Lead intoxication from ingestion of fishing sinkers: a case study and review of the literature. Clin Pediatr (Phila) 2008;47:66-70. CrossRef

12. Wu ML, Deng JF, Lin KP, Tsai WJ. Lead, mercury, and arsenic poisoning due to topical use of traditional Chinese medicines. Am J Med 2013;126:451-4. CrossRef

13. Hsiao CM. Dao of alchemy. Available from: http://taiwanpedia.culture.tw/en/content?ID=2081. Accessed 18 Sep 2013.

14. Lead and motor neurone disease. BMJ 1978;2:308. CrossRef

15. Kamel F, Umbach DM, Munsat TL, Shefner JM, Hu H, Sandler DP. Lead exposure and amyotrophic lateral sclerosis. Epidemiology 2002;13:311-9. CrossRef

16. Rademakers R, van Blitterswijk M. Motor neuron disease in 2012: novel causal genes and disease modifiers. Nat Rev Neurol 2013;9:63-4. CrossRef

17. Windebank A J, McCall JT, Dyck PJ. Metal neuropathy: peripheral neuropathy. 3rd ed. Philadelphia: WB Sanders; 1993: 1549-70.

18. Porru S, Alessio L. The use of chelating agents in occupational lead poisoning. Occup Med (Lond) 1996;46:41-8. CrossRef

Haemophagocytic lymphohistiocytosis (HLH) is characterised by fever, hepatosplenomegaly, central nervous system symptoms, cytopenia, coagulopathy, and lipid changes because of pathological immune activation, hypercytokinaemia and organ infiltration by phagocytosing histiocytes. Despite being an aggressive disease, effective treatment does exist. A treatment protocol was firstly designed in 19941 and later revised in 2004 by the HLH Study Group of the Histiocyte Society.2 Since the implementation of the treatment protocol of HLH 1994, its 5-year survival rate has improved from around 20% to more than 50%.1

Notably, HLH is comprised of two different conditions: familial/primary HLH (FHL) and secondary HLH (Table 1),3 with the former being an autosomal recessive condition. Five mutations that lead to FHL (Nos 1-5) have now been identified and the underlying genetic defect described in four. They are: PRF1, UNC13D, STX11, and STXBP2. For genetic defect in FHL1, the potential gene locus has been identified but not the specific genetic defect. The perforin gene (PRF1) mutation is the first genetic defect described in FHL (FHL2) and accounts for 20% to 50% of all affected FHL families identified in a Japanese study.4 Perforin is a soluble, pore-forming cytolytic protein synthesised in cytotoxic lymphocytes. This molecule plays a crucial role in regulating the access of granzymes to the cytosol of target cells, where they cleave key substrates to initiate apoptotic cell death. It is sequestered, along with granzyme serine proteases, in secretory cytotoxic granules. The PRF1 mutation results in reduction of perforin protein production and its cytotoxic function. This in turn impairs the control of lymphocyte homeostasis during immune responses and leads to hypercytokinaemia and continued expansion of populations of histiocytes and activated cytotoxic lymphocytes.5 Patients without an identifiable genetic cause but with a clear familial history of HLH are also classified as having FHL.

The differentiation of primary and secondary HLH is notoriously difficult. The only definite way is by genetic study to find the causative mutation. In Asia, the perforin gene mutation has mostly been identified in HLH patients in Japan.6 7 The case reported here is the first and the only HLH perforin gene mutation identified in Hong Kong.

The patient was the first child in a non-consanguineous family, with no history of previous unexplained deaths in the parents’ families. The child presented at 34 days of life with fever, hepatosplenomegaly, and pancytopenia in June 2009. The ferritin level was markedly increased to 18 513 (reference range [RR], 29-333) pmol/L and there was hypofibrinogenaemia with a level of 0.54 (RR, 1.85-3.83) g/L. Bone marrow examination showed features of haemophagocytosis. The diagnostic criteria for HLH were therefore met. Initial magnetic resonance imaging (MRI) of the brain and cerebrospinal fluid examination were normal. Virology investigations including serology for Epstein-Barr virus (EBV), human herpesvirus 6, herpes simplex virus, and cytomegalovirus were all negative. The patient was treated according to HLH 2004 protocol with dexamethasone, cyclosporine A, and etoposide. Her clinical condition deteriorated with severe metabolic acidosis and she underwent haemodialysis. She experienced persistent neutropenia after the first dose of etoposide. Repeat bone marrow examinations showed markedly depressed granulopoiesis with residual haemophagocytic activity. Further doses of etoposide were therefore withheld while dexamethasone and cyclosporine A were continued. The first course of chemotherapy was stopped after the 11th week of treatment. However, the patient had a relapse of HLH 3 weeks after stopping chemotherapy (manifesting as fever and hepatosplenomegaly). Repeat bone marrow examination confirmed the presence of haemophagocytic activity, for which treatment with dexamethasone, etoposide, and cyclosporine A was restarted. She developed progressive metabolic acidosis8 that was once again treated by haemodialysis. Her condition then became stabilised.

Since this patient presented at early age and had a recurrence after cessation of chemotherapy, she was suspected to suffer from FHL, for which the ultimate treatment is haematopoietic stem cell transplant (HSCT). Search for a related or unrelated donor was started while the patient continued to receive chemotherapy.

While waiting for the HSCT, the patient developed tremors of the lower limbs, and bilateral ankle clonus, limb spasticity and intermittent squints (with no definite visual fixation) were noted. The developmental age regressed from 8 to 3 months, whilst brain MRI revealed diffuse parenchymal and leptomeningeal enhancing lesions suggestive of lymphohistiocytic infiltration. Cerebrospinal fluid also showed presence of pleocytosis and a lymphohistiocytic infiltrate. The patient was diagnosed to have central nervous system involvement by HLH. Three doses of intrathecal chemotherapy with methotrexate 6 mg and hydrocortisone 8 mg were given over a 10-day period.

The patient received an unrelated double-unit cord blood transplant after conditioning with oral busulphan (23 mg/kg), etoposide (30 mg/kg), cyclophosphamide (120 mg/kg), and thymoglobulin (7.5 mg/kg). She had a neutropenic fever on post-transplant day 12. Donor cell engraftment was achieved on post-transplant day 16. Regrettably, she developed veno-occlusive disease causing hyperbilirubinaemia, fluid retention, progressive hepatosplenomegaly, and respiratory distress. The maximum bilirubin level was 209 μmol/L. Despite intensive care unit treatment, intubation, and positive pressure ventilation, the patient developed respiratory failure and died on day 45 after cord blood transplant. The parents refused a full postmortem. A postmortem liver biopsy showed marked sinusoidal dilatation and congestion with atrophy of central hepatocytes. These features were compatible with sinusoidal obstruction due to veno-occlusive disease.

Genetic analysis of the patient and the parents’ blood was performed, with the coding region of the perforin gene in exons 2 and 3 amplification by a polymerase chain reaction. This revealed a heterozygous one base pair deletion (65 delC) in exon 2 in the patient and her father. There was another mutation 853-855 del AAG in exon 3 of patient and her mother. Collectively, the patient had compound heterozygous mutations of the perforin gene, namely 853-855 del AAG and 65delC.

Previously we reported our seven consecutive cases of HLH encountered from 1991 to 2006.9 Since then, there had been four other patients. Apart from showing haemophagocytosis in bone marrow or lymph node or both (Table 2), all eleven patients had fever, splenomegaly, and cytopenia in at least two cell lines. They also had markedly elevated ferritin levels with or without a raised fasting triglyceride level or hypofibrinogenaemia. The standard diagnostic criteria for HLH were met in all patients. Another teaching hospital in Hong Kong reported nine cases from 1991 to 2006.10 The overall survival of all 20 patients was 58%.

Interestingly, EBV infection was confirmed (by immunoglobulin M vs EBV or EBV DNA) in 11 (55%) of the 20 patients. The overall survival of EBV-related HLH was 55%. In eight patients, their secondary HLH was related to malignant histiocytosis, Still’s disease, and anaplastic large cell lymphoma. Two patients had X-linked lymphoproliferative disorders.10 They all had primary EBV infection and one had died. The other patient received a mismatched unrelated cord blood transplant and had a full recovery without recurrence.

Four patients in our unit were investigated for perforin gene mutations; only one whom had an abnormality (compound heterozygous mutations in the gene). Hence this patient is the first reported case in Hong Kong with a perforin gene mutation causing FHL.

In Asia, most perforin gene mutations of HLH patients have been reported from Japan. Ueda et al6 reported five of 14 HLH patients with perforin gene abnormalities. The 1090-1091delCT and 207delC mutations of the perforin gene were frequently present in Japanese HLH patients. Ueda et al4 also reported a collaborative study which did not show PRF1 gene mutations from Korea (n=4), Malaysia (n=3), Hong Kong (n=2), Australia (n=1), and Taiwan (n=1). Lee et al11 from Taiwan reported 26 HLH patients; none of whom had PRF1, Mun12-4, STX11, or SH2D1A mutations. There was only one case report of a heterozygous PRF1 mutation (Arg390stop) in a young Taiwanese girl who presented with a panniculitis-like T-cell lymphoma and subsequently endured fatal HLH.12

Among the 20 patients in Hong Kong, only one had a PRF1 gene mutation and two had X-linked lymphoproliferative disorders. However, the actual rate of genetic abnormalities in HLH patients remains unknown as not all patients with HLH had genetic testing. This is partially due to inconsistent protocols for genetic investigations in this disease entity and inadequate laboratory support for genetic tests. Clearly, revision of the local clinical and laboratory service protocol is warranted, and more importantly, an international multicentre collaboration to improve immunological assessment and genetic analysis of HLH patients should be promoted.

1. Henter JI, Arico M, Egeler RM, et al. HLH-94: a treatment protocol for hemophagocytic lymphohistiocytosis. HLH study Group of the Histiocyte Society. Med Pediatr Oncol 1997;28:342-7. CrossRef

2. Henter JI, Home A, Aricó M, et al. HLH-2004: Diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer 2007;48:124-31. CrossRef

3. Freeman HR, Ramanan AV. Review of haemophagocytic lymphohistiocytosis. Arch Dis Child 2011;96:688-93. CrossRef

4. Ueda I, Ghim T, Peng LH, et al. Proceedings of the 2nd Congress Asian Society for Pediatric Research; 2006 Dec 8-10; Yokohama, Japan.

5. Usmani GH, Woda BA, Newburger PE. Advances in understanding the pathogenesis of HLH. Br J Haematol 2013;161:609-22. CrossRef

6. Ueda I, Morimoto A, Inaba T, et al. Characteristic perforin gene mutations of haemophagocytic lymphohistiocytosis patients in Japan. Br J Haematol 2003;121:503-10. CrossRef

7. Ueda I, Kurokawa Y, Koike K, et al. Late-onset cases of familial hemophagocytic lymphohistiocytosis with missense perforin gene mutations. Am J Hematol 2007;82:427-32. CrossRef

8. Hui WF, Luk CW, Chan WK, Miu TY, Yuen HL. Severe lactic acidosis in an infant with haemophagocytic lymphohistiocytosis. Hong Kong J Paediatr (New Series) 2012;17:183-9.

9. Chan JS, Shing MM, Lee V, Li CK, Yuen P. Haemophagocytic lymphohistiocytosis in Hong Kong children. Hong Kong Med J 2008;14:308-13.

10. Ho MH, Cheuk DK, Lee TL, Ha SY, Lau YL. Haemophagocytic lymphohistiocytosis in Hong Kong children have a wider clinical spectrum. Hong Kong Med J 2008;14:503-4.

11. Lee WI, Chen SH, Hung IJ, et al. Clinical aspects, immunologic assessment, and genetic analysis in Taiwanese children with hemophagocytic lymphohistiocytosis. Pediatr Infect Dis J 2009;28:30-4. CrossRef

12. Chen RL, Hsu YH, Ueda I, et al. Cytophagic histiocytic panniculitis with fatal haemophagocytic lymphohistiocytosis in a paediatric patient with perforin gene mutation. J Clin Pathol 2007;60:1168-9. CrossRef

In September 2010, a 40-year-old man presented to a general practitioner with multiple skin tags and acanthosis nigricans. He was noted to have acromegalic features, including prominent supraorbital ridge, prognathism, and spade-like hands. He was referred to Tseung Kwan O Hospital medical out-patient clinic. In the interim, he visited a private endocrinologist. Investigations demonstrated that his serum insulin-like growth factor 1 (IGF-1) level was markedly elevated, being 719 (reference range, 101-267) ng/mL, and his serum growth hormone (GH) levels were not suppressed following an oral glucose tolerance test (OGTT) with a trough GH level of 9.9 ng/mL, which confirmed the diagnosis of acromegaly. Magnetic resonance imaging (MRI) of the pituitary gland (Fig 1a and b) revealed a bulky pituitary gland with a 1.3 x 1.0 x 1.1 cm (transverse x height x anteroposterior dimensions) subtle roundish area in the central anterior part of the gland. Transsphenoidal resection of the pituitary macroadenoma was planned. However, preoperative chest X-ray (Fig 2a) showed a large mass at the right lower zone. Thus, the operation was cancelled and computed tomography (CT) thorax showed a well-defined lobulated mass (Fig 2b and c), measuring 8.6 x 7.4 x 7.1 cm (lateral x anteroposterior x craniocaudal dimensions), at the basal region of the right lower lobe. Fluorodeoxyglucose-positron emission tomography (FDG-PET) of the whole body suggested that the mass was consistent with a primary lung cancer; there were no intrapulmonary or distant metastases. Right lower lobectomy was performed by a private cardiothoracic surgeon in October 2010. Histology confirmed the tumour to be an atypical bronchial carcinoid. He was first seen by us in November 2012. Evaluation showed that his IGF-1 level and GH response after having an OGTT had normalised. Repeat MRI of the pituitary gland 17 months after the lobectomy (Fig 1c and d) showed that the gland had decreased in size compared with its earlier size and the previously noted structural lesion had vanished. In light of the co-existence of bronchial carcinoid and a history of a pituitary lesion, multiple endocrine neoplasia type 1 (MEN-1) syndrome was suspected, but genetic testing could not detect any mutations. Although growth hormone–releasing hormone (GHRH) level was not available, the patient most likely suffered from a GHRH-secreting bronchial carcinoid as suggested by the presence of a histologically confirmed bronchial carcinoid tumour, and normalisation of serum IGF-1 level and normal GH response following an OGTT upon complete removal of his lung tumour.

Acromegaly is due to sustained and unregulated hypersecretion of GH. It develops insidiously and progresses slowly, and typically remains undiagnosed for about 10 years.1 More than 95% of cases are caused by autonomous secretion of GH from anterior pituitary tumours and result in clonal expansion of somatotrophs. Less than 1% are due to ectopic GHRH production, with bronchial carcinoids being the most common cause (70%) followed by pancreatic islet cell carcinoids.2

Since the majority of bronchial carcinoids arise in the proximal airways, patients usually present with pulmonary symptoms,3 including cough, shortness of breath, wheeze, haemoptysis, chest pain, or recurrent pneumonia in the same pulmonary segment or lobe (due to bronchial obstruction). Although many of the tumours express immunoreactive GHRH, most patients with bronchial carcinoid are not clinically acromegalic. The first case of a bronchial carcinoid causing acromegaly was reported in 1958.4 Despite the large size and central location of his tumour, our patient did not have any chest symptoms; instead he came to medical attention because of prominent acromegalic features.

The clinical manifestations of acromegaly in patients with the ectopic GHRH syndrome are indistinguishable from those of any GH-secreting pituitary adenoma.5 Similarly, regardless of the cause, serum GH and IGF-1 levels are invariably elevated and GH levels fail to suppress (&lt1 ng/mL) during OGTT in all forms of acromegaly.6 No dynamic tests are helpful in differentiating the causes.7 Among all, plasma GHRH is the most precise and cost-effective test for the diagnosis of ectopic GHRH causing acromegaly. Plasma GHRH levels are usually elevated in patients with peripheral GHRH-secreting tumours, and are normal or low in patients with pituitary acromegaly.8 Regrettably, before the operation plasma GHRH level was not checked in our patient as this test was not available in most of the local hospitals. The presence of positive staining for GHRH could also provide direct evidence of the diagnosis.

Bronchial carcinoids are usually picked up easily on chest X-rays and by CT thorax. Compared with chest X-rays, CT delineates the extent of the tumour and its location better as well as the presence of any mediastinal lymphadenopathy. In our patient, the bronchial carcinoid was visualised by FDG-PET. However, FDG-PET yields conflicting results when it comes to identifying bronchial carcinoids, probably because of their small size and hypometabolic nature. In a retrospective review of 16 patients with surgically resected bronchial carcinoids, preoperative PET detected only 12 (75%).9 The use of other PET tracers, such as 11C-L-DOPA and 11C-5-hydroxytryptophan, improves the sensitivity for imaging neuroendocrine tumours.10 Approximately 80% and 60% of typical and atypical bronchial carcinoids express somatostatin receptors by immunohistochemistry, respectively. They may also be imaged with octreoscan.11 However, specificity is limited because scintigraphy is positive in many other tumours, and not all carcinoid tumours that express somatostatin receptors by immunohistochemistry test positive with octreoscan.

Pituitary gland MRI is necessary to verify the presence and size of a pituitary lesion, even when the diagnosis of ectopic GHRH syndrome has been established. In contrast to patients with classical acromegaly, no pituitary tumour but an enlargement of the sella is detected in the majority of such patients.12 The first MRI of the pituitary gland in our patient suggested the presence of an anterior pituitary macroadenoma, which is unexpected in patients with GHRH-secreting bronchial carcinoid. Thus, three other issues need to be considered. First, the co-existing carcinoid tumour and possible pituitary adenoma alerted us to the possibility of MEN-1. Second, the bronchial carcinoid might have metastasised to the pituitary gland. Third, the acromegaly really was due to the pituitary tumour producing excessive amounts of GH, and that its auto-infarction leads to normalisation of IGF-1, a normal GH response after an OGTT and shrinkage of the tumour on subsequent MRI. The absence of an MEN-1 mutation and hyperparathyroidism, and the resolution of pituitary lesion after lobectomy make the first possibility unlikely. Although we did not have any histology from the pituitary, again, disappearance of the lesion after the lobectomy also makes the second possibility unlikely. Regarding the third possibility, it cannot be proved or disproved in the absence of a plasma GHRH level and tumour histology. Nevertheless, we have to follow the patient closely to obtain the final answer.

Surgical resection of the bronchial carcinoids offers the best chance of cure, the prognosis of following resection of a typical carcinoid is excellent, with reported 5-year survival rates of 87% to 100%. While for atypical carcinoid, 5-year survival of 30% to 95% has been reported.3 13 Chemotherapy and radiotherapy are generally not effective. For those with non-resectable, disseminated tumours; who refuse surgery; or who are unsuitable because of medical co-morbidities, long-acting somatostatin analogues provide an effective option to control symptoms, and according to some studies, may also slow tumour progression.14

Ectopic GHRH acromegaly is so rare that routine screening would have a very low yield. Instead, clinicians should bear this diagnosis in mind, and search for an extrapituitary source of GH excess in those with unexpected clinical features (eg breathlessness, wheeze, or facial flushing), absence of a pituitary tumour on imaging, and the presence of tumours known to be associated with extrapituitary acromegaly. Measurement of plasma GHRH is the most cost-effective means of arriving at a diagnosis, but is not widely available. Chest X-ray, CT thorax and abdomen could be performed, if plasma GHRH testing is not available. A correct diagnosis is important, as the primary treatment for extrapituitary acromegaly entails surgical removal of the underlying tumour. Long-acting somatostatin analogues might be used to control symptoms, if resection is incomplete or not feasible.

1. Cordero RA, Barkan AL. Current diagnosis of acromegaly. Rev Endo Metab Discord 2008;9:13-9. CrossRef

2. Sano T, Asa SL, Kovacs K. Growth hormone-releasing hormone-producing tumors: clinical, biochemical, and morphological manifestations. Endocr Rev 1988;9:357-73. CrossRef

3. Skuladottir H, Hirsch FR, Hansen HH, Olsen JH. Pulmonary neuroendocrine tumors: incidence and prognosis of histological subtypes. A population-based study in Denmark. Lung Cancer 2002;37:127-35. CrossRef

4. Atmann HW, Schutz W. Uber ein knochenhaltiges Bronchuskarzinoid [in German]. Beitr Pathol Anat 1958;120:455-73.

5. Agha A, Farrell L, Downey P, Keeling P, Leen E, Sreenan S. Acromegaly secondary to growth hormone releasing hormone secretion. Ir J Med Sci 2005;173:215-6. CrossRef

6. Bonadonna S, Doga M, Gola M, Mazziotti G, Giustina A. Diagnosis and treatment of acromegaly and its complications: consensus guidelines. J Endocrinol Invest 2008;28(11 Suppl International):43-7.

7. Giustina A, Schettino M, Bodini C, Doga M, Licini M, Giustina G. Effect of galanin on the growth hormone (GH) to GH-releasing hormone in acromegaly. Metabolism 1992;41:1291-4. CrossRef

8. Mayo KE. Molecular cloning and expression of a pituitary receptor for growth hormone-releasing hormone. Mol Endocrinol 1991;6:1734-44. CrossRef

9. Daniels CE, Lowe VJ, Aubry MC, Allen MS, Jett JR. The utility of fluorodeoxyglucose positron emission tomography in the evaluation of carcinoid tumors presenting as pulmonary nodules. Chest 2007;131:255-60. CrossRef

10. Orlefors H, Sundin A, Garske U, et al. Whole-body (11)C-5-hydroxytryptophan positron emission tomography as a universal imaging technique for neuroendocrine tumors: comparison with somatostatin receptor scintigraphy and computed tomography. J Clin Endocrinol Metab 2005;90:3392-400. CrossRef

11. Granberg D, Sundin A, Janson ET, Oberg K, Skogseid B, Westlin JE. Octreoscan in patients with bronchial carcinoid tumours. Clin Endocrinol (Oxf) 2003;59:793-9. CrossRef

12. Losa M, Schopohl J, von Werder K. Ectopic secretion of growth hormone-releasing hormone in man. J Endocrinol Invest 1993;16:69-81. CrossRef

13. Asamura H, Kameya T, Matsuno Y, et al. Neuroendocrine neoplasms of the lung: a prognostic spectrum. J Clin Oncol 2006;24:70-6. CrossRef

14. Drange MR, Melmed S. Long-acting lanreotide induces clinical and biochemical remission of acromegaly caused by disseminated growth hormone-releasing hormone-secreting carcinoid. J Clin Endocrinol Metab 1998;83:3104-9. CrossRef

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

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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

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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