Genetics

Objective: To assess whether knowledge of insurance implications influenced uptake of genetic testing by participants in a research study of the causes of colorectal cancer.Design, setting and participants: Analysis of uptake of genetic testing by participants in the population-based Victorian Colorectal Cancer Family Study during two periods: from 1999 to 2003, when participants were not informed of any potential effect of genetic testing conducted during the study on their eligibility for new insurance policies; and from 2003 to 2006, when the protocol was changed to provide participants with information on the potential effect of genetic testing on insurance eligibility.Main outcome measure: Uptake of genetic testing for germline mutations in DNA mismatch repair (MMR) genes at a family cancer clinic.Results: The proportion of participants who declined genetic testing among those informed of insurance implications was more than double the proportion among those without this knowledge (29/59 [49%] v 9/47 [19%]; P = 0.002). This difference could not be explained statistically by adjusting for measured putative predictors.Conclusion: Identification of people with a mutation in an MMR gene has clinical importance, and such screening may be a cost-effective way to reduce the burden of colorectal cancer in the community. If people are choosing not to obtain genetic information because of how it will affect their eligibility for insurance, reforms to existing insurance practices are indicated.

Louise A Keogh BSc, MA, PhD · Christine M van Vliet BSc, MB BS, MPH · David M Studdert LLB, ScD · Judith A Maskiell BSc(Nursing), GradDip(BusMgt) · Finlay A Macrae FRACP, FRCP, MD · D James St John MB BS, MD, FRCP · Clara L Gaff BSc, FHGSA(GenCounsel), PhD · Mary Anne Young GradDip(FamilyTherapy), FHGSA(GenCounsel), MHSc(GenCouns) · Melissa C Southey BSc, PhD, GradDipLaw · Graham G Giles BSc, MSc, PhD · Doreen A Rosenthal BA, PhD · John L Hopper BSc, MSc, PhD · Mark A Jenkins BSc, PhD

One of the dilemmas in delaying sex-assignment surgery is the increased risk of gonadal malignancy Few areas of medicine are as controversial as the management of disorders of sex development (DSD). The use of the term DSD to describe patients born with ambiguous genitalia has undergone major change from older terms with negative connotations, such as “intersex”, “testicular feminisation” and “hermaphroditism”.1 Meanwhile, international debate continues about the ethics of performing genital surgery on affected infants and children. In fact, the debate has been raging for more than a decade between the medical profession and patient advocacy groups in Western countries, and has been documented by anthropologist Katrina Karkazis in a recent book.2 A long-term outcome study of 50 patients aged 18–32 years who had been treated in Melbourne when they were children showed that mental and physical health outcomes were as good for most of the DSD patients as for those in two control groups; however, there was a small minority of patients whose gender identity as adults was a source of such profound discomfort that they felt compelled to undergo treatment to change it.3 Clearly, this is unsatisfactory, and management practices have been reviewed internationally by clinicians looking for ways of minimising the risk of making such mistakes about gender assignment. The main problem relates to feminising genitoplasty (Box), which involves the removal of phallic erectile tissues and skin that cannot be replaced. This type of operation is considered appropriate for 46,XX girls with congenital adrenal hyperplasia (Box), who rarely identify as male when they are adults if they are treated with appropriate hormones to maintain androgen suppression from soon after birth and throughout childhood.4 However, feminising genitoplasty is much more of a problem in patients with a Y chromosome. For example, in one study of 14 adult patients with genetically confirmed partial androgen insensitivity who were treated at Johns Hopkins University in the United States as children, 25% experienced gender dysphoria (Box) as adults, and a small number wanted to undergo sex change surgery.5 Although policy changes are still being discussed, it seems likely that fewer and fewer XY patients with frankly ambiguous genitalia due to DSD will have feminising genitoplasty and be raised female. The option to assign a gender but postpone surgery until the child is able to give consent has been strongly advocated in some quarters,6 but has not gained much traction because of concerns that children might suffer psychological harm if left with ambiguous genitalia. In 2008, clinicians from Melbourne’s Royal Children’s Hospital, recognised for their expertise in the management of DSD, were required to meet representatives of the Victorian state Justice Department. They were asked to respond to a proposal — advanced by an advisory committee representing the interests of the gay, lesbian, bisexual, transsexual and intersex communities — that doctors wanting to perform surgery to treat ambiguous genitalia in children too young to consent on their own behalf should have to seek approval from the Family Court of Australia on a case-by-case basis. Also in 2008, the Australian Human Rights Commission decided to initiate a public inquiry into the same question, and circulated a draft discussion paper called Genital surgery for babies born intersex to health professionals for comment. Thus, in Australia as elsewhere, the arm wrestle between medical professionals and patient advocacy groups continues. What has largely been missing from the debate is recognition of the fact that surgery forms a necessary part of the risk management strategy for preventing gonadal malignancy. In any DSD associated with a Y chromosome, there is an increased risk of germ cell cancer,7 especially when the testes are intra-abdominal (the risk of seminoma in partial androgen insensitivity is 50% for an intra-abdominal testis) or when there is gonadal dysgenesis. In this issue of the Journal, a salutary case report by Parker and colleagues8 reminds us of the need to be mindful of this risk, and also to take a long-term view of risk. If the intra-abdominal gonad in the patient described had been removed at the initial surgery, he would never have needed to fear this tumour. It had not been removed because, by today’s standards, he had been inadequately investigated in the past, and therefore the intersex condition was not recognised. The trend for surgeons to recommend male-sex rearing for greater numbers of children with DSD could also mean greater reluctance to remove testes that pose a significant risk of cancer on the grounds that physiologically useful hormone secretion might be retained. It is therefore imperative that a risk management strategy be prepared for each patient. This would mandate: educating parents and patients about risk; removing all intra-abdominal gonads that cannot be brought down into the scrotum; regular clinical and ultrasound surveillance of scrotal gonads with removal of any that contain suspicious lumps; biopsy of testes after the onset of puberty, looking for early signs of malignant change; and effective communication between paediatric and adult care-providers at the time of transition. It is also important for all children identified as having DSD to be referred to a centre of excellence where they will be seen by paediatric endocrinologists, surgeons and other health care professionals with expertise in the field and who recognise the importance of a multidisciplinary team approach.9 Case conferences about patients diagnosed as having a DSD in adult life would be enhanced if paediatric specialists in DSD were asked to comment. Of equally great importance is the need for an accurate aetiological diagnosis wherever possible. At the moment, about 40% of patients with 46,XY forms of DSD are left without a precise diagnosis.10 The application of microarray (gene chip) technology,11 which is available in Australia, is an exciting and promising step forward in identifying genetic mutations. In this technique, samples of very large numbers of genes are arranged in a regular pattern on a solid surface or membrane, which is then incubated with DNA from a patient. Alterations in known (and even unknown) genes are rapidly detected by studying patterns of matches and mismatches. The current challenge for researchers is to develop new tools, such as microarray technology, that will lead to gene discovery and to better methods of screening patients for mutations in all the known genes. Glossary of terms relating to disorders of sex development DSD: Disorders of sex development, previously known as intersex. Congenital conditions in which development of the chromosomal, gonadal or anatomical sex is atypical. Feminising genitoplasty: Surgery carried out to give genitalia that were originally ambiguous a more female appearance. Usually involves clitoral reduction (removal of erectile tissue) and surgery to create a vaginal opening separate from the urethra. Congenital adrenal hyperplasia: A genetic disorder caused by a deficiency of the enzyme 21-hydroxylase in the adrenal cortex, and the commonest adrenal disorder of childhood. Cause of virilisation in an affected female fetus. Partial androgen insensitivity: An X-linked genetic disorder causing ambiguous genitalia in 46,XY individuals. Caused by a lack of androgen receptors in androgen target tissues, such as genital skin. Gender dysphoria: Mental distress caused by unhappiness with one’s own sex and the desire to be identified as the opposite sex.

Garry L Warne MB BS, FRACP · Jacqueline K Hewitt MB BS

Disorders of sex development (DSD), previously termed intersex, are uncommon, and are usually, but not always, diagnosed at birth. Issues of gender assignment, psychosexual development and the potential for malignant change in a dysgenetic gonad need to be considered. Here, we report a rare presentation of advanced malignancy in an abdominal gonad associated with the formation of a uterus in an adult male with a previously undiagnosed DSD. Clinical recordA 59-year-old man presented with haematuria and gynaecomastia, and was subsequently found to have a large pelvic mass. Examination of his left breast identified a 3–4 cm mobile mass. Ultrasonography of the breast confirmed a mass, but could not distinguish between glandular tissue and a breast tumour. Computed tomography (CT) of the abdomen and pelvis showed a 10 cm complex pelvic mass that was thought to extend to the seminal vesicles. A core biopsy showed mixed cytological and immunohistochemical features of a possible granulosa cell tumour, but the precise diagnosis was limited by the small amount of material present for examination. A provisional diagnosis of gonadal malignancy in a dysgenetic gonad was considered. A bone scan was negative and a thoracic CT scan showed three 5–7 mm lesions in the lung fields, suggestive of possible metastases. His prostate-specific antigen (PSA) level was very low (0.01 ng/mL), and tests for testicular tumour markers were negative (alpha-fetoprotein, 3 ng/mL; human chorionic gonadotropin, < 5 U/L). Other biochemical tests showed the following levels: serum oestradiol, 372 pmol/L (male reference range [RR], < 160 pmol/L); testosterone, 1.1 nmol/L (RR, 8–38 nmol/L); follicle-stimulating hormone, < 0.1 U/L (male RR, 1.0–9.2 U/L); luteinising hormone, 2.1 U/L (male RR, 2–8 U/L); prolactin, 385 mU/L (RR, 0–500 mU/L). The patient had a history of penoscrotal hypospadias, cryptorchidism and right inguinal hernia that were surgically repaired in infancy. The hypospadias was corrected at 20 months of age. He had a right inguinal hernia repair at 3 years of age and, at that time, was found to have an undescended right testis that was treated by orchidopexy. The patient’s parents were told that the left testis was removed during this procedure. He was married with no children, and we do not know whether the issue of fertility had ever been questioned. He was noted to have minor obstructive urinary symptoms at 47 years of age that were thought to be caused by a urethral stricture secondary to his previous hypospadias repair. He re-presented 8 years later with a bloody urethral discharge that settled after treatment with antibiotics. On rectal examination at age 55, he had a small prostate and his PSA level was low (0.02 ng/mL). At that time, an intravenous pyelogram showed normal kidneys, ureters and bladder. At cystoscopy, he was found to have a mid-urethral stricture from his previous hypospadias repair, with a number of hairs present in the skin at the stricture site. The hairs were thought to be from graft skin used to repair his hypospadias, and this was felt to be the cause of his bleeding. He re-presented with intermittent urethral blood loss 2 years later, at 57 years of age, and repeat cystoscopy again showed a mid-urethral stricture that was treated by dilatation. At 59 years of age, a laparotomy was performed and the patient was found to have a large left adnexal mass (Box 1, A) and a normally formed uterus. The mass was removed and a subtotal hysterectomy performed as there was no distinct lower margin of the cervix (Box 1, B). The patient was informed of the operative findings and the diagnosis of intersex. Peripheral blood karyotyping showed a 46,XY genotype. The histopathology of the gonadal tumour showed a sex cord-stromal tumour of indeterminate differentiation (Box 2). The histopathology of the uterus showed simple endometrial hyperplasia. The patient was treated with postoperative chemotherapy, but died 18 months later. DiscussionDisorders of sex development (DSD) are rare in the general population, and comprise a wide variety of clinical, anatomical and genetic problems. Most patients with DSD are diagnosed at birth, but diagnosis may not be made until childhood, adolescence or, more rarely, in adulthood, as in our case. This case shows how incomplete initial assessment and treatment of a neonate with abnormal genital development, and failure to recognise this rare disorder in adulthood can have tragic, adverse health consequences. An international consensus statement on intersex disorders and their management proposed the term “disorders of sex development” to describe congenital conditions with atypical development of chromosomal, gonadal and anatomical sex.1 It was considered that previous terminology, such as hermaphroditism, was controversial, pejorative to patients and confusing. DSD are a heterogeneous group of conditions, all of which interfere with normal sex differentiation in the embryo and fetus. The incidence of DSD in the population is estimated to be one in 5500.2 Patients with DSD can present at birth with ambiguous genitalia, apparent female or apparent male genitalia. Patients with apparent female genitalia can have an enlarged clitoris, posterior labial fusion or an inguinal / labial mass. Patients with apparent male genitalia can have non-palpable testes, micropenis, isolated perineal hypospadias or hypospadias with undescended testes. Congenital adrenal hyperplasia is the most common cause of ambiguous genitalia in the newborn period.2 The birth of an infant with ambiguous genitalia requires a strategy of clinical, hormonal, genetic, molecular and radiographic investigations to determine the aetiology and to plan a therapeutic approach.3 Clinical and diagnostic evaluation protocols are available.2 Although most DSD are diagnosed in the neonatal period, a wide variety of clinical presentations have been reported in childhood, adolescence and adulthood. Later presentations include previously unrecognised genital ambiguity, inguinal hernia in a girl, delayed or incomplete puberty, primary amenorrhoea, virilisation in a girl, breast development in a boy and gross haematuria in an adolescent male.1,2 A wide variety of clinical presentations have been reported in adults with undiagnosed DSD. These include psychiatric disturbances,4 short stature, infertility,4,5 lower abdominal pain6 or haematuria. Our patient presented with intermittent haematuria that was initially investigated with cystoscopy and an intravenous pyelogram. A computed tomography urogram with intravenous contrast has now replaced intravenous pyelography as the standard investigation of the urinary tract in patients with haematuria. If this investigation had been performed at the time of his earlier presentations, a pelvic mass may have been identified and further investigated. Recent advances in molecular genetics have increased our understanding of both normal and abnormal sex development. The bipotential gonad develops at the urogenital ridge between 5 and 6 weeks’ gestation and a number of genes involved in testis determination are expressed at this stage.7 The first histological evidence of testis formation occurs at 7 weeks’ gestation. Leydig cell synthesis of testosterone mediates the development of the vas deferens, epididymis and seminal vesicles, and conversion of testosterone to its more potent form, dihydrotestosterone, masculinises the external genitalia to form a penis and scrotum. Sertoli cell synthesis of anti-Müllerian hormone prevents the Müllerian ducts from developing into a uterus and fallopian tubes.7,8 The development of internal reproductive organs is controlled by the ipsilateral gonad, and prenatal male sex development is finally completed at term by descent of the testes into the scrotum.7 Our patient appears to have produced enough testosterone from the small right testis to promote penile development, albeit with hypospadias. The left gonadal dysgenesis appears to have resulted in inadequate anti-Müllerian hormone production, allowing a uterus to develop. The risk of malignancy in dysgenetic gonads is significantly increased in some patients with DSD.8 The presence of the SPY gene on the GBY region of the Y chromosome is a prerequisite for malignant transformation.9 Tumours can arise in any of the gonadal cells or their precursors.8 Precursor lesions for the development of cancers occur as carcinoma-in-situ in testicular tissue and gonadoblastoma in undifferentiated gonadal tissue. A number of malignant tumour types may occur in dysgenetic gonads.8,10 These include germ cell tumours and sex cord-stromal tumours. Patients presenting with abdominal tumours in dysgenetic gonads in adulthood provide histopathologists with complex diagnostic dilemmas. Histopathological examination of the tumour in our case showed mixed elements, with cells resembling ovarian follicles, testicular tunica, granulosa cells, Sertoli cells and Leydig cells. No germ cell components were identified. The final consensus was a diagnosis of malignant sex cord-stromal tumour of indeterminate differentiation. It is likely that the oestrogen-secreting gonadal tumour had been present for some years in our patient, resulting in endometrial hyperplasia. The uterus is likely to have been connected to the urethra, with endometrial bleeding causing the apparent haematuria. Persisting elevated levels of oestrogen would also have caused gynaecomastia, which resolved after surgical removal of the tumour. Serum follicle-stimulating hormone was likely to have been suppressed by the elevated level of oestradiol, and a normal level of serum luteinising hormone and low level of testosterone are consistent with the history of incomplete external genital development and infertility. Clinicians managing neonates with cryptorchidism and proximal hypospadias should have a high index of suspicion of an underlying DSD.11 Once a DSD is identified, a series of investigations should be initiated with the aim of making a specific diagnosis.7,12 Physical examination and imaging studies should be performed to attempt to locate inguinal or abdominal gonads so that surgical removal can be considered in patients thought to be at high risk of subsequent malignancy.2,10 The sequence of events in our case highlights the diagnostic difficulties that can occur in patients who present in adulthood with unsuspected gonadal dysgenesis. 1 Macroscopic photographs of the left adnexal mass A: Macroscopic photograph of malignant sex cord-stromal tumourdisplaying widespread necrosis and haemorrhage. B: Macroscopic photograph of bisected uterus with a thickened endometrium. 2 Histopathology of the gonadal tumour Microscopic photograph of gonadal tumour. The better differentiated areas of the tumour showed nests of cells with peripheral palisadingand small Call–Exner body-like structures (arrows), suggesting granulosa cell differentiation. Microscopic photograph of gonadal tumour. Also present were areas showing primitive sex cord-like structures (white arrow), lumened tubules lined by eosinophilic epithelial cells and adjacent clustered Leydig cells (black arrow).

Jim L Parker BMed, DRACOG, FRANZCOG · Deborah L Ekman BAppSc · Lawrence J Hayden FRCS

In June 2008, the ABC screened a television documentary involving a couple who decided to have an additional child in the hope of obtaining umbilical cord blood to treat their daughter who had leukaemia. The couple conceived naturally, meaning that there was a one in four chance that their child would be suitably matched. They seemed to be unaware of technologies that, if successful, could provide a near certainty that the next child would be a matched “saviour sibling”. This story raises questions about whether clinicians have an obligation to discuss emerging and morally contentious treatment options. Ignorance of technology, assumptions about availability, and medical assessment of burdens and benefits may affect attitudes towards treatment options, but they do not justify non-disclosure of information.

Kimberly A Strong BSc, GradDipGenCouns

Objective: To explore the experiences of families with a baby with Down syndrome at the time of diagnosis, and their preferences for information and support in the early period after diagnosis.Design, setting and participants: A qualitative, interview-based study of 18 families living in Victoria with a child with Down syndrome born between 2002 and 2004 who had not been diagnosed with the syndrome before birth. Interviews were transcribed verbatim and interpretive content analysis was undertaken.Results: Parental coping with the unexpected diagnosis of Down syndrome in their infant was influenced by the time interval between birth and disclosure of clinical suspicion of Down syndrome, the level of certainty of the attending physician at the time of disclosure, and the time interval between disclosure of clinical suspicion and confirmation of karyotype. Initial uncertainty and a delay in the diagnosis were detrimental to parental coping, as was premature communication of the news. Perinatal complications increased parental anxiety regarding their child’s condition and future. Individual communication style of midwives and physicians was a powerful predictor of parental adaptation. Parental needs for support and information were facilitated through normalising postnatal care, ensuring privacy, and providing early access to peer support and up-to-date written information. Many parents would have appreciated access to a liaison worker.Conclusion: The experiences of parents in this study provide practice points for improving postnatal care with minimal changes to formal service systems.

Evelyne E Muggli MPH · Veronica R Collins PhD · Catherine Marraffa FRACP, FRCPCH

To the Editor: We report the case of a 41-year-old woman who sought medical opinion about an unpleasant body odour, first noticed when she was 7 years old. After experiencing ridicule, distress, shame, anxiety and low self-esteem during her school years, she first consulted a doctor about the problem at the age of 17 years, then again 2 years later, followed by a further four doctors over the next 20 years. All dismissed her concerns, and she was repeatedly told that she had a hygiene neurosis. Investigations and treatments during this time included being “sniffed”, vaginal swabs and vaginal cauterisation. Finally, a general practitioner referred her to a dermatologist, who consulted a microbiologist, and the diagnosis of trimethylaminuria (TMAU), or fish malodour syndrome, was confirmed by urinalysis. Now having a name for her condition, she found an Internet-based support foundation and referred herself for genetic counselling. TMAU is caused by an enzyme deficiency due to mutations in the flavin-containing mono-oxygenase 3 (FMO3) gene,1 resulting in excess excretion of trimethylamine in urine, sweat and breath. It is diagnosed by clinical symptoms and urine analysis.2 The characteristic body odour resembling rotting fish can be intermittent, variable and influenced by diet, hormones and medications. Restriction of choline- and carnitine-rich dietary precursors (eg, fish, eggs, soybeans, peas) is difficult to maintain and effective in only 25% of patients.2 Acid soaps and body lotions can often reduce the odour.3 The metabolic and clinical manifestations of TMAU are generally regarded as benign, as there is no associated organ dysfunction. This designation, and the fact that the condition is often unrecognised by doctors, can have important ramifications including missed or delayed diagnosis.4 Affected individuals experience shame and embarrassment, fail to maintain relationships, avoid contact with people who comment on their condition, and are obsessive about masking the odour with hygiene products and even smoking. The malodorous aspect can have serious and destructive effects on schooling, personal life, career and relationships, resulting in social isolation, low self-esteem, depression, paranoid behaviour, and suicide.4, Psychosocial problems resulting from delayed diagnosis, body odour and the lack of cure are considerable, making this a far from “benign” disorder. Recognition of TMAU as a significant clinical entity and increased understanding of the issues patients face are needed. Awareness of the typical patient history would facilitate prompt metabolic diagnosis and pre-empt some of the associated psychosocial sequelae. Referral of patients for genetic counselling enables short-term psychosocial support and family cascade genetic testing. Consultation with a metabolic clinic for dietary management may also be beneficial.

Helen Mountain · Joanna M Brisbane · Amanda J Hooper · John R Burnett · Jack Goldblatt

Long (or short) QT syndrome is life-threatening, not as rare as once thought, and treatable if diagnosed “The fault dear Brutus is not in our stars, but in ourselves”. Through the lens of molecular medicine we are now beginning to see those faults more clearly. We can now increasingly understand why some apparently healthy children and young adults die without warning. The long-QT syndrome is foremost among responsible causes, and is known to be the consequence of mutations in genes encoding ion channel function.1 Originally an esoteric condition of great rarity, its prevalence is now estimated as one in 2000.2 Life depends on the continuous sequence of depolarisation and repolarisation in our heart cells. The QT interval is the time from the onset of depolarisation (the q wave) to the end of repolarisation (completion of the T wave) and is best measured over several R-R intervals in leads II or V5. It is not a value physicians ordinarily pay much attention to when they read an electrocardiogram (ECG; Box). Nor is it always easy to measure. Exactly where the T wave merges with the isoelectric baseline is subject to individual interpretation and hence error.3 It also varies with heart rate, requiring normalisation for comparison, and is longer in women than men. But it is important. The long-QT syndrome is essentially an autosomal dominant condition in which sudden death is commonly linked to situations of increased adrenergic activity, such as exercise and emotion, but also occurs at rest and during sleep.4 Seizures are common and easily lead to misdiagnosis. Although mutations in at least 10 genes have been found, long-QT syndromes 1 (LQT1), 2 (LQT2) and 3 (LQT3) constitute 95% of genotyped cases. LQT1 and LQT2 involve decreased function and hence diminished current flow in the slow and fast repolarising potassium currents, IKs and IKr, respectively, while LQT3 involves an increase in slow sodium current during the plateau phase of the action potential. The net effect is prolongation of the action potential and, most importantly, an increased dispersion of recovery times in different myocardial cells. This increased dispersion allows re-entry to occur with potentially fatal ventricular tachycardia and fibrillation, which manifests clinically as fainting and sudden death. LQT1 is the most common type, and events are typically triggered by exercise, including swimming, and emotion.5 The child found unconscious at the bottom of the swimming pool may well have LQT1.6 Events may occur with exercise or at rest in LQT2 and also, characteristically, with loud noises such as being awakened by a telephone call.7 LQT3 has been linked to death during sleep or inactivity, with a lower likelihood of events, but increased mortality. What are the implications for physicans and general practitioners? Fainting is common, most often vasovagally mediated, and benign. How do we decide otherwise? The key is to be mindful of possible long-QT syndrome when checking the history; fainting or a seizure during exercise, or when upset or angry, and premature death (including drownings or accidents) in family members should ring alarm bells and trigger detailed exploration of the family history, close scrutiny of ECGs and appropriate referral. In such settings, a corrected QT (QTc) interval > 0.45 seconds in males and > 0.47 seconds in females makes the diagnosis virtually certain. With diagnosis comes the dual responsibility of triaging individual patient risk and screening the wider family. The most important factor determining individual risk is the length of the QT interval — long intervals (QTc ≥ 0.5 seconds) equal high risk.8 Knowledge of the genotype is also predictive. β-Blockers are generally first-line treatment, although in genotyped individuals, evidence is lacking for a protective action in LQT3. Modifying risk by avoidance of competitive sport and QT-prolonging drugs (see http://www.qtdrugs.org) is important. Implantable defibrillators are appropriate in high-risk patients, but decisions about prophylactic implantation in intermediate-risk patients must balance the reduction in probability of sudden death against the not inconsiderable morbidity of life-long device therapy in young people. As well as personal history and ECG, family screening should include genetic testing if it is available. When a functionally important mutation is uncovered, testing of family members will disclose up to a third of individuals whose QT intervals are normal, and yet who carry the mutation.9 However, a genotypic diagnosis is possible in only two-thirds of clinically certain cases. Continuing research may diminish this gap. The establishment of registries, such as presently exist in New Zealand (http://www.cidg.org), facilitates surveillance of widely dispersed families and aids ongoing research. While the risks of QT prolongation are now well established, attention has recently been drawn to excessively short QT intervals. In a small number of families identified to date, a QTc interval of < 340 milliseconds has been also associated with a family history of sudden death.10 The first two syndromes described (short-QT syndromes 1 and 2), show a gain of channel function for IKr and IKs, the mirror opposite of the corresponding LQT2 and LQT1 with loss of function in those same channels. Although five short-QT syndromes have been recognised already, it nonetheless seems unlikely that they will rival the long-QT syndrome in prevalence. In conclusion, cardiac repolarisation is a complex interplay of ionic currents precariously maintaining stability. Dramatic progress has been made over the past 50 years in recognising, deciphering and predicting the clinical risk of the long-QT syndrome. Yet this knowledge counts for little if we fail to identify and protect at-risk individuals. Perhaps you will take a closer look at the QT interval in your next patient presenting with “just” another fainting attack. Electrocardiogram showing a markedly prolonged QT interval. The long ST segment is suggestive of long-QT syndrome 3.

Warren M Smith MB BS, FRACP

To the Editor: A recent editorial in the Journal suggested that blood could be sent overseas for genetic testing for maturity onset diabetes of the young (MODY).1 We are pleased to be able to point out that genetic testing, including clinical and laboratory support with full gene sequencing for both MODY1 and MODY3 and for neonatal diabetes (mutations in SUR1 and Kir6.2), is available in Australia. Testing for MODY2 and for a number of other disorders of the pituitary–adrenal and pituitary–gonadal axis in children is also available. We are happy to receive specimens and referrals from clinicians who would prefer to use an Australian clinical laboratory accredited by the National Association of Testing Authorities. More information is available via: http://www.mater.org.au/Home/Services/Pathology.aspx.

Mark F Harris · Ivan N McGown · David M Cowley

Objective: To audit the clinical indications for HFE gene mutation testing in a consecutive series of requests.Design: Retrospective audit of reasons prompting 187 HFE test requests received between June 2003 and June 2005, by examination of the request form, hospital notes (when available) and, when required, information from the referring doctor.Setting: A tertiary care public teaching hospital laboratory, Perth, Western Australia.Main outcome measures: Reasons prompting requests for HFE genotype testing and compliance with accepted clinical indications (biochemical evidence of iron overload on repeated samples, or a first-degree relative with either haemochromatosis or a C282Y mutation).Results: Insufficient clinical details in requests prevented the inclusion of interpretive comments in HFE genotype reports in 70 of 187 cases (37%). Re-evaluation after collation of the missing details for all but seven requests revealed that 103 of the 180 auditable requests (57%) had been prompted for reasons other than biochemical evidence of iron accumulation or family history.Conclusions: A substantial proportion of HFE genotype test requests are made for inappropriate reasons. Clinical practice could be improved by educating doctors on the practical utility of this genetic test and by laboratories taking steps to secure the clinical information needed to include appropriate interpretive comments in their reports.

Melissa J Gillett FRCPA, FRACP · Cyril D Mamotte PhD · David Ravine MD, FRACP, FRCPA · Samuel D Vasikaran MD, FRCPA

To the Editor: The recent article by Miller and colleagues regarding adverse drug events (ADEs) in general practice highlights the high frequency and considerable morbidity associated with ADEs in the general community.1 The authors identified recognised side effects, drug sensitivity, and allergy as responsible for most ADEs. The contribution of the patient’s genotype to drug response, via altered metabolism or responsiveness to pharmaceuticals, is increasingly recognised as potentially responsible for a significant proportion of ADEs. The science of determination of the genetic contribution to an individual’s response to drug action is referred to as pharmacogenomics,2 and represents a potentially beneficial diagnostic tool to aid in the prevention of ADEs. Treatment with warfarin, one of the most frequently prescribed drugs in Australia, has been estimated to account for up to15.1% of all severe ADEs, manifest as minor and major bleeding.3 We have recently determined the presence, frequency and laboratory sequelae of genetic variants (single nucleotide polymorphisms) in two genes responsible for the metabolism (cytochrome P450 2C9 [CYP2C9]) and potency (vitamin K epoxide reductase complex, subunit 1 [VKORC1]) of warfarin4 in an Australian population. In our study of 120 patients in an anticoagulation clinic, the frequencies of allelic variants of the CYP2C9 and VKORC1 genes responsible for altered warfarin activity were 31%5 and 59% (unpublished data), respectively, in keeping with previously published studies.4 Detection of these variants was associated with increased induction international normalised ratio (INR) readings compared with controls, and reduced overall warfarin requirements.6 These findings support previous studies,7 and suggest that genotype determination may be of benefit in identification of patients with increased sensitivity to empiric induction phase warfarin dosing schedules. This may allow for a reduction of induction doses of warfarin, decreasing the risk of excessive INR and bleeding sequelae, commonly observed with induction of warfarin treatment. Furthermore these benefits may aid in reduced time to stabilisation. Additional cost–benefit analysis8,9 will enable determination of the economic viability of genotype determination as an adjunct to management of warfarin dosing. The high population frequency of genetic variants associated with warfarin response emphasises the significant contribution genetic factors can play in patient reaction to drugs and highlights their involvement as potential causes of ADEs.

Keith A Byron · Anthony E Dear

The Time Eaters We have to live here for ever. Think of what for ever means! Back to Methuselah G B Shaw What sets the internal clock of species, regulates the rapid beat of rodents, slow pendulum stroke of pachyderms? A dog is born, dies in the breath of a boy’s years. I searched skeletal remains of millennial men, an archeology of bone. The cyclic give and take of osteoblast, osteoclast confirmed the biblical chronologies, as arboreal rings mark seasons of Sequoias. In ancient DNA, a taxonomy of telomeres, I found the answer — a retroviral presence, the chronophage, passed through generations. In the Cave of the Patriarchs, a wind out of Eden touched my face. Quarantined in their Garden, Adam and Eve lived eternal lives — caught mortality, the slow descent to untimely death as they departed into the world.

Richard Bronson MD

Molecular genetics can facilitate a successful switch to oral diabetes therapy The increase in type 1 and type 2 diabetes in childhood has been well documented worldwide and in Australia.1,2 In addition, the separate entity of monogenic diabetes is increasingly recognised in paediatric diabetes, and now encompasses neonatal diabetes mellitus and maturity onset diabetes of the young (MODY)3 (Box 1). Monogenic diabetes is defined as diabetes caused by a single gene defect. A diagnosis of monogenic diabetes should be considered in a child who is diabetes-associated-autoantibody negative, is diagnosed with diabetes in the first 6 months of life, has a parent with diabetes, and/or is not markedly obese. Although uncommon — its frequency is estimated to be 1%–3% of all childhood diabetes3 — the clinical relevance of this condition is that at least some of those affected (in particular, those with MODY1 and MODY3) can achieve very good diabetes control with sulfonylurea rather than insulin therapy. Neonatal diabetes mellitus presents in the first 6 months of life with signs of hyperglycaemia — polyuria, dehydration, failure to thrive and, in many, frank diabetic ketoacidosis. Diabetic ketoacidosis is an important diagnosis to consider in an infant who presents critically unwell because the clinical picture may mimic sepsis. While the reported incidence of neonatal diabetes mellitus is one in 500 000 newborns,4 the estimated incidence is thought to be a lot higher, and it may be the cause of some unexplained infant deaths. About half of affected patients will have transient neonatal diabetes mellitus, where insulin treatment can be discontinued within a median of 3 months (although diabetes mellitus may recur in the second or third decade of life). In contrast, patients with permanent neonatal diabetes mellitus have, until recently, required insulin therapy for life. The revolution in patient management we describe here is due to molecular genetic analysis of the ATP-sensitive potassium (KATP) channel of the pancreatic beta cell (Box 2). Sulfonylureas have traditionally been used to treat type 2 diabetes mellitus. They act by binding the sulfonylurea receptor (SUR1), which closes KATP channels, thereby stimulating endogenous insulin production from the pancreatic beta cell. Gloyn et al demonstrated that some patients with Kir6.2 potassium channel activating mutations secreted insulin in response to the intravenous sulfonylurea tolbutamide.5 Subsequently, the Neonatal Diabetes International Collaborative Group conducted a trial of glibenclamide, an oral sulfonylurea, in 49 patients with Kir6.2 mutations. This trial included two Australian centres, with three children — one white and two of Middle Eastern ethnicity. An impressive 90% of the trial patients were successfully switched from insulin to glibenclamide.8 Importantly, the responsiveness in vitro of mutant ATP channels to tolbutamide was proportionate to the patient’s response to glibenclamide. This enables a degree of predictability of whether a patient is likely to successfully switch from insulin to oral therapy. Not only was oral therapy welcomed by families of patients, but the switch from insulin resulted in significant improvement of metabolic control, with glycated haemoglobin levels dropping from 8.1% to 6.4% after 12 weeks of treatment.8 Insulin response to oral glucose load was increased in those tested. Continuous glucose monitoring has also shown fewer fluctuations in postprandial glucose,9 which families report improves the child’s general wellbeing. However, the story is not all rosy — some patients with Kir6.2 activating mutations known to have poor in-vitro response to tolbutamide may not be able to switch to oral therapy. It is therefore important to determine the exact genetic mutation involved, so that families can be counselled about the chances of a successful switch. In our experience, such counselling was helpful in lessening the disappointment when a 7-year-old girl with a Kir6.2 mutation, who had presented with ketoacidosis at 7 months of age, remained insulin-dependent despite maximal glibenclamide dose. The diagnosis of neonatal diabetes mellitus should be considered in any critically ill infant, and the International Society for Pediatric and Adolescent Diabetes recommends that all infants who develop diabetes mellitus in the first 6 months of life be tested for a genetic mutation in the KATP channel.10 DNA from peripheral blood can be sent to a diabetes research laboratory in Exeter in the United Kingdom for testing (see http://www.diabetesgenes.org). To date, 20 Australian children, who had been insulin-dependent from less than 6 months of age, have been genotyped. Seven tested positive for mutations in Kir6.2 and three for mutations in SUR1 (Professor Andrew Hattersley, Peninsula Medical School, Exeter, UK, personal communication), and some have gained insulin independence. While molecular genetics can now help classify and facilitate management of childhood diabetes, regardless of the type of diabetes (type 1, type 2, or monogenic), all children who present with severe fasting hyperglycaemia and ketoacidosis will initially require insulin therapy to reverse the metabolic abnormalities. 1 Classification of primary diabetes mellitus in children Type 1 diabetes is characterised by the presence of diabetes-associated autoantibodies (islet cell, insulin, glutamic acid decarboxylase, and tyrosine phosphatase). A number of children with type 1 diabetes may be obese at diagnosis. Type 2 diabetes is characterised by obesity, negative antibodies and raised C-peptide levels. It is more common in non-white people than type 1 diabetes. Comorbid obesity can make the distinction between these two types of diabetes difficult. Monogenic diabetes is caused by a single gene abnormality. Maturity onset diabetes of the young (MODY) 1 and MODY3 are due to transcription factor mutations. Children with monogenic diabetes are not generally obese. Some children with monogenic diabetes present in the neonatal period with ketoacidosis. 2 Subunit structure of the ATP-sensitive potassium (KATP) channel of the pancreatic beta cell* The KATP channel consists of four sulfonylurea receptor (SUR1) subunits and four potassium channel (Kir6.2) subunits. Closure of the KATP channel is required for glucose-stimulated insulin secretion from the pancreatic beta cell. Conversely, opening of the KATP channel inhibits insulin secretion. Inactivating mutations of genes encoding both SUR1 (ABCC8) and Kir6.2 (KCNJ11) subunits keep the channel closed and are known to cause uncontrolled insulin secretion, resulting in congenital hyperinsulinism. It was hypothesised that activating mutations of these genes would keep the KATP channel open and cause permanent neonatal diabetes mellitus (PNDM). In 2004, Gloyn et al reported six novel heterozygous mutations in 10 of 29 patients with PNDM, including a 5-year-old Sydney girl who had been treated with insulin from 6 weeks of age.5 Subsequently, Proks et al reported a patient with activating mutations of ABCC8,6 and Babenko et al reported ABCC8 mutations in two of 29 patients with PNDM and seven of 44 patients with transient neonatal diabetes mellitus.7 KATP channels are also found in skeletal muscle and neurones throughout the brain, and some patients with Kir6.2 activating mutations have extrapancreatic features — motor skill and language delay, muscle contractures, epilepsy, and dysmorphic features — leading to the description of a new syndrome, known as DEND (Developmental delay, Epilepsy, Neonatal Diabetes) syndrome. In-vitro studies of mutant KATP channels have shown a correlation between the degree of KATP channel insensitivity and severity of the clinical phenotype.5 * Adapted from: Sperling MA. ATP-sensitive potassium channels — neonatal diabetes mellitus and beyond [editorial]. N Engl J Med 2006; 355: 507-510. PIP2 = phosphatidyl-inositol-4,5-bisphosphate.

Shubha Srinivasan MB BS, MRCP, FRACP · Kim C Donaghue MB BS, PhD, FRACP

Clinical record A 44-year-old man underwent coronary artery bypass surgery in 2004. He had been in satisfactory health as an adult apart from hypertension, for which he was receiving treatment. He ate a normal diet, including dairy products, meat and other high protein food. At age 44 years, he developed acute central chest pain while exercising at a gymnasium. An angiogram showed coronary artery occlusions. Forty-eight hours after successful coronary artery bypass surgery, during which time he received intravenous sodium but not glucose, he felt unwell, and the following day he became delirious. Computed tomography of the brain, initially reported as appearing normal, was later thought to show cerebral oedema. Plasma ammonium level was 110 μmol/L (reference range [RR], 10–50 μmol/L), but the timing of this sample was unclear. Wilson’s disease was initially considered because of elevated liver enzyme levels, but serum copper and ceruloplasmin levels were not measured. Eight days after the bypass surgery, a urine sample was sent to the NSW Biochemical Genetics Service, for metabolic screening. This showed gross elevations in glutamine and orotic acid (orotic acid, 14.8 μmol/mmol creatinine; RR, 1.23 μmol/mmol creatinine), indicating a likely diagnosis of ornithine transcarbamylase (OTC) deficiency. Plasma glutamine level was 3527 μmol/L (RR, 385–862 μmol/L). Plasma tyrosine and methionine levels were also moderately elevated, which was consistent with liver dysfunction. Plasma citrulline level was mildly elevated at 57 μmol/L (RR, 10–45 μmol/L), but this finding was difficult to interpret in the light of the other elevated amino acid levels. (Citrulline level is very low in neonatal OTC deficiency, but not necessarily low in late-onset phenotypes.) The patient was by then gravely ill, requiring maximum life support, and under consideration for liver transplantation. His condition progressively deteriorated despite introduction of intravenous sodium benzoate and l-arginine, and life support was withdrawn. He died 8 days after surgery. The diagnosis of OTC deficiency was subsequently confirmed by mutation analysis which showed hemizygosity for the c.622G>A (p. A208T) mutation in exon 6 of the OTC gene (as males have only one X chromosome, they are said to be hemizygous with respect to X-linked genes). Cascade testing of family members (Figure) showed that the patient’s mother (person II-2) was a carrier, heterozygous for the c.622G>A (p. A208T) mutation, while his asymptomatic brother (III-2) was hemizygous for the same mutation. Random biochemical testing of the brother showed normal levels of plasma glutamine, citrulline, arginine and urinary orotic acid. No information was available on causes of death of first-generation relatives. Past history revealed that, at age 7 years, the patient had an episode of acute encephalopathy following a 2-month history of intermittent nausea, vomiting and frontal headache. There was no preceding febrile illness, intercurrent infection or history of trauma. The symptoms reappeared after a 2-week period of apparent recovery. On admission to hospital, he was drowsy, disorientated and restless, but quickly settled and made a satisfactory recovery. No haematological or biochemical results were evident in his medical records. He was discharged with a diagnosis of viral meningitis/encephalitis. We report a 44-year-old man who presented with fatal hyperammonaemia after coronary artery bypass surgery. He had previously been asymptomatic, apart from a possible episode of unrecognised hyperammonaemia in childhood. The diagnosis of ornithine transcarbamylase (OTC) deficiency was made too late for successful intervention. Inborn errors of metabolism are frequently unrecognised or diagnosed late in adults. OTC deficiency is the most common disorder affecting the urea cycle. In New South Wales, the incidence is of the order of one in 70 000 births.1 It is an X-linked disorder leading to potentially lethal hyperammonaemia. The clinical severity ranges from acute neonatal hyperammonaemic coma to symptom onset at any time from infancy to adulthood, depending on environmental triggers and residual enzyme activity.2 The timing of episodes in late-onset OTC is dictated by environmental factors that increase nitrogen turnover, including dramatic increase in protein intake, medications affecting protein catabolism, viral illness or other generalised stress, rapid weight loss, and poor nutritional intake.3 Lessons from practice Inborn errors of metabolism are frequently unrecognised or diagnosed late in adults. Postoperative catabolism with insufficient calorie intake may unmask previously asymptomatic, but potentially lethal, inborn metabolic errors. Patients with acute onset of unexplained neurological or psychiatric symptoms need urgent metabolic investigation, including measurement of plasma ammonia level, to exclude metabolic causes. The first step in ureagenesis is the production of carbamyl phosphate from ammonium and bicarbonate. OTC then catalyses the biosynthesis of citrulline from ornithine and carbamyl phosphate. Thus, a deficiency of OTC leads to accumulation of ammonia and glutamine (the major extrahepatic source of ammonia for ureagenesis), and a reduction in citrulline. The accumulating carbamyl phosphate enters the pyrimidine synthetic pathway, resulting in increased excretion of orotic acid. The missense mutation p. A208T, replacing alanine with threonine at codon 208 of exon 6 in the OTC gene, has been previously reported in a late-onset OTC patient we investigated,4 and in others.5-7 This group showed an extremely wide phenotype, ranging from encephalopathy at age 4 months7 to no symptoms at age 97 years.5 Our patient developed hyperammonaemia following postoperative catabolism, caused by surgical stress and fasting with inadequate calorie supply from intravenous fluids pre- and postoperatively. In addition, a high nitrogen load from bleeding sites, tissue trauma or tissue protein breakdown could have overwhelmed urea synthesis and promoted the excessive ammonia production. OTC deficiency is not the only inborn error of metabolism that can result in fatal postoperative decompensation. Another example is medium-chain acyl-CoA dehydrogenase deficiency, which appears more prevalent, with several recorded cases (eg, Raymond et al8). Other mild fatty-acid oxidation defects and, perhaps, intermittent maple syrup urine disease could behave similarly. Death during an initial episode seems frequent in patients with late-onset OTC deficiency, as lack of familiarity with the disorder in the adult setting delays diagnosis and appropriate treatment.9-11 Treatment of hyperammonaemia is well established, and includes aggressive calorie support to counteract catabolism, early use of intravenous sodium benzoate as an ammonia sink, and intravenous arginine.2 It was unfortunate that no objective evidence was gathered on the cause of the patient’s episode of encephalopathy during childhood. While the cause could have been viral encephalitis, the clinical course, and appearance of the cerebrospinal fluid on microscopy and the brain on computed tomography did not strongly support this diagnosis. Assessment of plasma ammonia level would most likely have led to the diagnosis of OTC deficiency. Establishment of the correct diagnosis in the patient led to the finding that his mother and brother were also affected, enabling them to be advised about precautions. The genetic implications for the family’s younger generation were not critical, as the proband had no children, and his brother’s children were both male and could not have inherited their father’s X chromosome (Figure). This case illustrates the difficulty of diagnosing late-onset OTC. The X-linked inheritance may be obscured, even when more than one family member is affected, as some patients remain asymptomatic. Patients with acute onset of unexplained neurological or psychiatric symptoms need urgent metabolic investigation, including measurement of plasma ammonia level, to exclude metabolic causes. Cascade testing of family members The G-to-A base change in exon 6, which results in an amino acid substitution from alanine to threonine at position 208 (p. A208T), was identified in DNA from the blood of the patient with late-onset ornithine transcarbamylase deficiency (person III-1) and his brother (III-2). Their mother (II-2) was found to be heterozygous for the p. A208T mutation.

Mary Anne Chiong MD · Bruce H Bennetts PhD · Simone I Strasser MD, FRACP · Bridget Wilcken FRACP, FHGSA

Objective: To assess the evidence of an association between periconceptional folic acid (FA) supplementation or fortification of foods with FA and the risk of twinning, using the Food Standards Australia New Zealand (FSANZ) framework for assessing evidence when substantiating nutrition, health and related claims on foods.Data sources: The Cochrane Library Database, MEDLINE, MEDLINE in Process, EMBASE, PubMed National Library of Medicine, and CINAHL were searched to identify systematic reviews and primary intervention and observational studies published from 1 July 1994 to 7 July 2006.Study selection: One prospective and five retrospective cohort studies that assessed the rate of twinning in populations exposed to FA through supplementation, and six retrospective registry-based cohort studies examining twinning rates after fortification of foods with FA.Data extraction: Two reviewers appraised eligible studies and evaluated data independently.Data synthesis: The best maximal risk estimates of twinning after FA supplementation were an adjusted odds ratio (adjOR) of 1.26 (95% CI, 0.91–1.73) for preconceptional supplementation and dizygotic twinning and an adjOR of 1.02 (95% CI, 0.85–1.24) for overall twinning. Data from four FA fortification studies in the United States that allowed for calculation of an annual percentage increase showed a maximal annual increase in twinning rates of 4.6%.Conclusions: Overall, under the FSANZ framework, there is possible evidence for a relationship between periconceptional FA intake and increased twinning. To support this tentative relationship, more well designed, long-term follow-up studies are needed in places where fortification with FA has been introduced, focusing on dose–response and obtaining accurate data on infertility treatments.

Evelyne E Muggli MPH · Jane L Halliday PhD

Health services research is important to ensure continued best quality of care, but often uses data obtained without explicit consent for this purpose. Obtaining consent may be difficult for many reasons, but excluding individuals may introduce biases that alter the significance of studies. Approval by ethics committees of a waiver of the need for consent allowed our study to proceed and provide evidence that has led to the implementation of a population-based screening policy for the prospective detection of hereditary non-polyposis colorectal cancer. This screening policy has resulted in more cases being detected routinely with better management for affected patients and their at-risk families. A need for consent would have prohibited this study, and the development of a more efficient screening policy could have been delayed for several more years. Ethics committees can effectively manage the need to uphold basic ethical principles without unnecessarily impeding socially useful research. Committees need to be familiar with the guidelines approved under sections 95 and 95A of the Privacy Act 1988 (Cwlth) in addition to the National Health and Medical Research Council National statement on ethical conduct in research involving humans.

Nikolajs Zeps PhD · Barry J Iacopetta PhD · Lyn Schofield MPH · Jillian M George BHealthSci, RN · Jack Goldblatt MB ChB, MD, FRACP

Family, adoption and twin studies demonstrate that many adult psychiatric disorders, including schizophrenia, major depression and bipolar disorder, have a clear genetic component. The aetiology of psychiatric disorders is a complex combination of both genetic and environmental components. While potential susceptibility genes for psychiatric disorders have been identified, interaction with the environment is a crucial component in disease development. Pharmacogenetics and genetic testing have the potential to play key roles in the future of clinical psychiatry. At present, an increased risk of psychiatric disorders can be identified through a detailed family history. The empirical risk of developing a disorder has been determined for many psychiatric disorders and can be used as a general guide. Genetic counselling can extend and enhance patient care by providing information to patients about the complexities of inheriting psychiatric disorders and the associated risks of recurrence. The genetic counselling process can facilitate informed decision making, alleviate misconceptions and reduce stigma through an improved understanding of the genetic cause of psychiatric disorders, and offer support to patients and their families.

Melissa K Hill PhD · Margaret Sahhar BA, DipSocStuds

Stem cells. Controversy at the frontiers of science. Elizabeth Finkel. Sydney: ABC Books, 2005 (vi + 282 pp). ISBN 0 7333 1248 9. There has been no more controversial area in medical science in recent years than the discovery that embryonic and adult stem cells have the potential to generate a range of different tissues, possibly even organ repair and regeneration. As the field has evolved at a blistering pace, several important strands can be discerned. Firstly, the science, while complex, is of great interest to the lay reader. For the public to adequately understand and appraise the importance and potential of these new developments, it is crucial that the current state of scientific development be explained in readily understandable terms. Secondly, the area of stem cell therapy, especially embryonic stem cells and their development, raises many complex ethical issues divisive in our society. Thirdly, Australian scientists have played a major role both in the development of in-vitro fertilisation, the precursor to the embryonic stem cell age, and in the development of embryonic and adult stem cells as potential treatments of the future. Elizabeth Finkel, a distinguished science journalist with a strong background in embryology, has written an intriguing account of the stem cell story. She has brought together all three strands into a very readable account of the field. On one level, for those interested in human stories in science, this is a very good read with great human achievement and political drama. It is also one of the best accounts of the scientific achievements with stem cells to date and of the basis for arising ethical issues. Finkel makes no secret that she is an enthusiast for developing stem cell science as a viable treatment, with both embryonic and adult stem cell research. In some of the examples she covers, most notably Parkinson’s disease, she may overstate the current state of success of stem cell therapies just a little, but does so in an attempt to give a balanced story. One of her key conclusions, that scientific facts must be presented and discussed dispassionately, and that ethical considerations and viewpoints should not be allowed to bias interpretation and presentation of the science to the general public, is a pivotal one in all aspects of science. Stem cell therapy, whether derived from embryonic or adult stem cells, has a great way to go before we will know for sure whether it will be a viable treatment in a major sense for human illness. Elizabeth Finkel’s book makes clear that it is an area of very considerable promise, and I commend this riveting account to all who have an interest in the area. Edward ByrneDean of Medicine, Nursing and Health Sciences, Monash University, VIC Competing interests: Professor Byrne is the current Dean of Medicine at Monash University, where much of the stem cell work in this book was carried out. “Stem cells” won the Science Writer’s Award in the 2005 Queensland Premier’s Literary Awards

Edward Byrne

Some boys conceived through artificial techniques may inherit their fathers’ subfertility Clinical assessment of couples unable to conceive naturally often identifies causative or contributory factors associated with the male partner. Male infertility affects one in 20 men, accounts for a third of all infertility, and is a cofactor in over half of assisted reproductive technology (ART) treatments worldwide.1 Primary spermatogenic failure (SgF, also termed idiopathic infertility) accounts for more than half the cases, yet, in most of these cases, its cause is unknown.1 In clinical practice, classification of SgF is based on semen parameters (describing combinations of poor sperm number, motility or function) and reflects an ignorance of the pathogenesis.2 However, recent research has determined that up to 15% of SgF is related to at least six known Y chromosomal deletions, with implications for genetic testing, counselling, assisted reproduction and even subsequent male offspring conceived by ART. Spermatogenesis is a complex process of cell division and structural modification involving the coordinated expression and interplay of many gene products. Recent data point increasingly towards a genetic basis for SgF. In particular, deletions of the Y chromosome — called microdeletions — are the most significant recognised cause of SgF in otherwise healthy men.3 The Y chromosome is 60 megabases (Mb) in size, and comprises a short arm (Yp) that encodes the male sex-determining gene Sry, and a long arm (Yq) (Box). Of the 27 Y chromosome genes identified, nine are located on Yp and the remaining 18 on Yq.4 Twelve of the 18 Yq genes are expressed in a testes-specific manner and are vital for normal sperm production.4 Y chromosome microdeletions in fact involve substantial DNA deletions within the Yq region, ranging from 1.6 to 14.5 Mb and, depending on the deletion type, result in the loss of specific combinations of spermatogenic genes. Accordingly, men with Yq microdeletions are often (but not always) infertile, but many can still father children through intracytoplasmic sperm injection (ICSI),5 using the few viable sperm present in semen or mature spermatids isolated directly from the testis.6 An association between Y chromosomal deletions and infertility was first reported in 1976 by Tiepolo and Zuffardi,7 who detected large Yq deletions in six azoospermic men by routine karyotyping involving chromosomal banding. They proposed that an azoospermia factor (AZF) region was associated with spermatogenesis. Extensive physical, functional and genetic analyses of the Y chromosome8,9 have now identified three AZF regions (AZFa, AZFb and AZFc), which encode spermatogenic genes such as USP9Y, RBMY1, and BPY2 and DAZ (Deleted in AZoospermia) (Box). DNA sequencing of the Y chromosome has identified unique structural features such as large palindromes (DNA sequences that read the same in both directions) that encompass highly repetitive DNA elements.4 Homologous recombination, involving elimination of one repetitive sequence at the expense of another, is believed to be the underlying mechanism that accounts for the random appearance of de novo AZF microdeletions in men. Interestingly, the fathers and brothers of men with Yq microdeletions usually have non-deleted Y chromosomes and normal sperm counts, indicating that these deletions are spontaneous events. The reason for the appearance of Yq microdeletions in some men is not known. We speculate that these deletion events occur during gametogenesis or early preimplantation development and may involve a deficiency in enzymes responsible for normal DNA repair. Up to 15% of men with SgF and sperm densities below 5 million/mL have AZF deletions.3 Variation in the reported incidence of Yq deletions in infertile men has been attributed to factors such as patient selection criteria, the molecular test format, and the propagation of specific Y chromosome types (called haplotypes) within population groups that have different susceptibilities to deletion events. The three identified AZF regions contribute to six different Yq deletion types: AZFa, AZFb, AZFc, AZFbc, AZFabc,8 and the gr/gr subdeletion9 within AZFc (Box). There is no clear relationship between genotype and spermatogenic phenotype, but some generalisations can be made. Most microdeletions (59.6%) involve the AZFc region3 and are associated with the histological appearance of hypospermatogenesis, which is characterised by a reduction in germ cell number, mature elongated spermatids in some or all tubules, and low sperm densities ranging from 5 million/mL to azoospermia.10 Less common Yq deletions involve AZFb (15.8%), AZFbc (13.6%), AZFa (4.9%) and AZFabc (< 1%) regions; such men are often azoospermic and have more severe spermatogenic pathologies, such as arrested germ cell development or the Sertoli-cell-only syndrome. In 6% of cases, Yq deletions that involve regions outside of the three AZF regions have been identified in men with spermatogenic failure.3 Recent investigations of the Y chromosome have identified several smaller deletions within the AZFc region. For example, we have found that one such deletion, called gr/gr,9,11 is more prevalent than AZFc deletions in severely oligospermic or azoospermic men (4.7% v 2.2%, respectively).12 However, we also found gr/gr deletions at a similar frequency in oligospermic men (sperm densities, 5–40 million/mL). Interestingly, gr/gr deletions have also been found in some fertile men who have a particular Y haplotype.13 Thus, gr/gr deletions are relatively independent of sperm density, but significantly associated with infertility. At this stage, the gr/gr deletion appears to be a risk factor for infertility rather than a definitive cause of SgF. The variable sperm parameters observed in men with AZFc and gr/gr deletions could be due to a number of factors, including molecular heterogeneity of the deletion or functional compensation of the lost DAZ gene by its gene homologue DAZLA on chromosome 3. We are currently involved in an international effort to define more precise correlations between sequence variants of these deletions and semen parameters. Vertical transmission of AZFc Yq deletions from infertile men to their sons via ICSI14 and natural conception13 has been reported, the latter underscoring that male fertility is possible even at low sperm output. We have generated a DNA database of more than 150 infertile men and their ICSI-conceived sons, and have identified and mapped the AZFc deletions in three Y-deleted men: in all cases, the same deletion was transmitted to the sons without expansion to other AZF regions.14 Furthermore, analysis of a larger panel of Y chromosomal markers located throughout the AZF and surrounding regions has not identified de novo Yq deletions in 100 ICSI-conceived sons tested to date, indicating that ISCI treatment is not a risk factor for the generation of Yq deletions. However, it seems very likely that these ICSI-conceived boys with AZFc deletions will be subfertile and will need close review as they reach sexual maturation and proceed into adulthood with aspirations for fatherhood. The European Molecular Genetics Quality Network (http://www.emqn.org) has established guidelines for Yq deletion testing and provides an important quality assurance function.15 These laboratory guidelines have been widely adopted and have led to standardisation of Yq testing. The test is based on the analysis of a large panel of conserved molecular markers or genes located within and outside the AZF regions, using multiplex polymerase chain reaction (PCR) on peripheral blood genomic DNA. The pattern of these markers determines the Yq deletion type. Because of the inherent instability of the Y chromosome, it is likely that new Yq microdeletions will be identified and associated with SgF. High-resolution microarrays for chromosome screening will enable further investigation of the Y chromosome in fertile and infertile men, and microarray-based testing may replace multiplex PCR as the gold standard for Y chromosome testing in the future. Given the relatively high prevalence of Yq deletions, most andrology and infertility centres now routinely offer Y chromosome testing to men with severe SgF, especially before ART treatment. Yq deletions have important implications for infertile couples, and genetic counselling following testing is recommended. There are several considerations that support routine assessment of Yq deletions. Firstly, a positive test will provide a firm diagnosis of the man’s problem, which, for some couples with longstanding infertility, can help resolve stress, blame or feelings of guilt. Secondly, knowledge of the type of Yq deletion may assist the clinician in determining the best ART treatment. For example, a Yq microdeletion involving the AZFa or AZFb regions carries a poor prospect of sperm retrieval, even with testicular biopsy,16 thereby questioning the value of this approach and raising donor sperm treatment for discussion. Thirdly, couples should be offered this information, as they must understand that their male offspring will almost certainly be subfertile and require reproductive monitoring from the time of sexual maturation. As the natural history of SgF is poorly understood, it seems wise that consideration be given to sperm storage, as these young men born by ICSI may move from oligospermia to azoospermia before seeking fatherhood. Lastly, infertile men are known to be at increased risk of androgen deficiency17 and testicular neoplasia;18 however, whether the subgroup with Yq deletions have a greater risk also requires careful monitoring. Following genetic counselling about their Yq deletion, most couples still proceed with in-vitro fertilisation using either the male partner’s sperm or donor sperm.19 In a small number of cases, couples have used preimplantation genetic diagnosis to select female embryos for transfer,19 in an attempt to avoid passing on the genetic abnormality to their children. The relationship of Y chromosome deletions and other genetic lesions to male infertility will continue to be an active area of interest. Given the widespread use of ICSI to resolve male infertility, it is important that this research is translated rapidly and appropriately into clinical practice, and that prospective couples are provided with essential information that allows them to be fully informed when making this crucial life decision. The human Y chromosome A: Normal Y chromosome showing AZF regions and representative spermatogenic genes. B: Different Y chromosome deletion types. Dotted lines indicate the deleted regions.

David S Cram PhD · Elissa Osborne PhD · Robert I McLachlan PhD, FRACP

Marfan syndrome (MFS) is a multisystem disorder of connective tissue that is inherited in an autosomal dominant fashion, and results from mutations in the FBN1 gene on chromosome 15. Diagnosis is challenging as it requires definition of diverse clinical features and input from a variety of specialists. Genetic testing of FBN1 is time consuming, expensive and complex, and may not solve the diagnostic dilemma. Failure to make a diagnosis or making an inappropriate diagnosis of MFS has social, lifestyle and medical consequences for the individual as well as the family.

Kim M Summers BSc(Hons), PhD · Jennifer A West RN · Madelyn M Peterson BPharm, MA(Ethics) · Denis Stark MB BS, FRCS, FRACO · James J McGill MB BS, FRACP · Malcolm J West MB BS, FRACP, PhD

To the Editor: This question was recently posed by Massie and colleagues in an editorial in the Journal.1 We have experience in providing cystic fibrosis (CF) carrier testing in pregnancy and in those planning pregnancy. In 1998, we ran a 12-month pilot program, the Double Testing Program, offering ΔF508 carrier testing to couples attending the John Hunter Hospital, Newcastle, NSW, for nuchal translucency screening. Of 491 participants, 84% chose CF carrier testing; 23 carrier–non-carrier couples were identified, and no carrier–carrier couples. A postnatal questionnaire compared knowledge of CF, anxiety levels and perceptions of the service between non-carrier couples and couples in which one partner was a carrier.2 Both groups were very satisfied with the service, with no increased levels of anxiety. The second initiative has been to offer CF carrier testing to clients attending the “drop-in” clinic at Hunter Genetics, Newcastle. This clinic has been available for 10 years and provides genetic counselling for clients referred by their general practitioners for pre-pregnancy or pregnancy counselling. Over the past 3 years, there have been 560 occasions of service with CF carrier testing in 499 individuals. Indications for testing include pregnancy screening or a family history of CF. With couples, both are tested for ΔF508, and, if one partner is identified as a carrier, the other partner is tested for 28 other mutations. The area health service, Hunter New England Health, meets the cost of the service and testing — $100 per couple for the ΔF508 test, and $250 for the full mutation screen. Of the 499 individuals, 65 (13%) were found to be CF carriers; after excluding those with a family history of CF, the carrier rate was 5.8%. Twenty carrier–non-carrier couples and one carrier–carrier couple with a family history of CF were identified. Benefits included enabling the carrier–carrier couple to know their 1 in 4 risk of having a child with CF. Most women who have a child with CF want to avoid having further affected children, and most who have a subsequent pregnancy choose prenatal diagnosis.3 Cascade testing can be offered to carrier families. Non-carrier couples can be reassured that they have a low risk. The uptake of CF couple screening is low, given that there are over 3500 births annually at the John Hunter Hospital. The main obstacles are lack of awareness and costs. CF tests are not covered by public health funding or by private health insurance, and this issue needs urgent attention. We believe that GPs are best placed to offer CF couple testing. Testing could be incorporated into routine first trimester pregnancy care. We can provide a distance CF learning package, pamphlets and informed consent material to those interested.

Louise M Christie · Elvira M Zilliacus · Angela J Ingrey · Gillian Turner

New tumour testing methods can improve the accuracy of diagnosis Although a strong genetic predisposition to colorectal cancer (CRC) is rare, it is important because of its large contribution to CRC diagnosed before the age of 50 years and because mortality from CRC can be reduced by appropriate management. There are currently limitations in determining whether a case of CRC is “sporadic” or whether it might be related to an inherited predisposition. However, Australian research that examines the utility of new diagnostic tools to help diagnose genetic tendency to CRC may be showing us a way forward.1,2 What is hereditary non-polyposis colorectal cancer?The two best-characterised high-risk inherited syndromes are familial adenomatous polyposis and hereditary non-polyposis colorectal cancer (HNPCC) — sometimes known as Lynch syndrome. Both are inherited as autosomal dominant traits. Familial adenomatous polyposis can usually be readily diagnosed on the basis of clinical findings alone, when an individual develops CRC at a relatively young age on a background of colorectal adenomatous polyposis (mostly with more than 100 adenomas). HNPCC cannot usually be readily diagnosed. HNPCC accounts for about 1%–4% of all CRC, but up to 10% in patients younger than 50 years at diagnosis. It is caused by a germline (heritable) mutation in one of a family of genes known as the DNA mismatch repair genes: hMLH1, hMSH2, hMSH6 and hPMS2.3 HNPCC is characterised clinically by an early age of onset of CRC, a predisposition for proximal colonic cancers, and a tendency to develop multiple CRC, along with an increased risk of some extra-colonic malignancies, including cancers of the uterus and ovary, stomach, small bowel, biliary tree, ureter, renal pelvis, pancreas and brain. The family history is often complex. What are the difficulties in diagnosing HNPCC?Traditionally, HNPCC has been suspected in families where the family history of cancer meets the modified Amsterdam criteria.4 These require a family to have at least three close relatives in two generations diagnosed with cancer of the colon, rectum, endometrium, small bowel, ureter or renal pelvis, with at least one diagnosed before age 50 years. One problem is that not all “Amsterdam positive” families will be proven to have HNPCC and, conversely, some families with proven HNPCC do not meet these criteria. Family history alone is not enough — a diagnosis of “suspected HNPCC” may be based on verified clinical and pathological information from the family pedigree, but the diagnosis of HNPCC is ultimately confirmed by the demonstration of a family germline mutation in one of the mismatch repair genes. This is done by taking blood from one of the affected family members and searching the mismatch repair genes to determine whether a causative mutation can be found. However, a mutation cannot be found in every family, as mutations may be missed or mutations may be present in other genes that are not yet identified. This means that if the family history is strong (Amsterdam positive) and the genetic test (mutation search) fails to identify a mutation in an affected family member, the test result should be considered “inconclusive” and all relatives remain at potentially high risk. Only when a causative mutation in one of the mismatch repair genes has been identified can other at-risk adult family members be offered “predictive” genetic testing to determine their risk. Importantly, those found not to carry the family mutation should be considered at the average population risk for cancer, and they (and their offspring) can be spared additional cancer screening and concern. At present, many families with a family history that meets or approaches the Amsterdam criteria are offered this expensive approach to germline genetic testing; few are found to have proven HNPCC. How are these difficulties being addressed?The accurate detection of HNPCC has improved recently for a number of reasons. The first is an increasing awareness of family history as a risk factor for CRC. Clinical practice guidelines assist in estimating CRC risk based on family history.5,6 For those with a more complex family history, referral to a family cancer clinic may be appropriate. These clinics have been developed to provide cancer risk assessment, surveillance, and prevention strategies, with genetic counselling and testing when appropriate. Finally, we now have improved diagnostic tools, including molecular testing of tumours for microsatellite instability and immunohistochemical staining of tumour tissue for mismatch repair proteins, which can be used to help investigate a history of “suspected HNPCC”. Additional molecular tumour tests are in development. Molecular testing for microsatellite instabilityMicrosatellite instability is one of the hallmarks of HNPCC-related cancers and is due to unrepaired mutations in repetitive sequences of DNA known as microsatellites. CRCs that occur in patients with HNPCC tend to be right-sided, mucinous, poorly differentiated and characterised by the presence of tumour infiltrating lymphocytes. On molecular testing, these tumours show high levels of microsatellite instability. The Bethesda guidelines7 were developed to provide criteria for testing tumours for microsatellite instability in an individual affected family member to assist in the diagnosis of HNPCC. These guidelines suggest that all people with CRC diagnosed before the age of 50 years should have microsatellite instability testing to look for possible HNPCC. However, another limitation presents itself. Although high levels of microsatellite instability are seen in most HNPCC-related tumours, microsatellite instability is not exclusive to HNPCC. Some people develop sporadic cancers with high levels of microsatellite instability, unrelated to an inherited defect in mismatch repair; around 10%–15% of sporadic colorectal cancers will exhibit high levels of microsatellite instability. This tends to occur in older women with right-sided cancers, often mucinous, with poor glandular differentiation (as per HNPCC), but with no associated HNPCC family history. In patients with sporadic CRC with high levels of microsatellite instability, the defect in mismatch repair is not heritable and occurs only in the tumour as a result epigenetic silencing of the promoter region of hMLH1. It does not alter the risk of CRC in offspring, so it is important to differentiate this from HNPCC. Tumour BRAF gene mutations occur in most of these sporadic cancers, but virtually never in HNPCC-associated cancers with high levels of microsatellite instability. The presence of a BRAF gene mutation in a tumour with high levels of microsatellite instability makes it more likely to be a sporadic CRC; however, the use of tumour testing for BRAF mutation is speculative at this stage and it is not yet readily available. Immunohistochemical stainingAnother approach used to improve the accuracy of diagnosis of HNPCC is the use of immunohistochemical staining in suspected cases. Immunohistochemical staining uses antibodies to the proteins encoded by the four relevant mismatch repair genes (hMLH1, hMSH2, hMSH6 and hPMS2) to test for the expression of these proteins in tumour tissue. Absence of mismatch repair proteins is often seen in HNPCC and may be specifically related to the gene in which a germline mutation may subsequently be found; that is, the absence of hMSH2 protein in cancer cells usually indicates a germline mutation in hMSH2. However, once again, this absence of staining is not specific to HNPCC — loss of expression of hMLH1 due to somatic inactivation of hMLH1 does occur in sporadic cancers. How is Australian research contributing in this field?Southey et al have recently conducted an analysis of the use of tumour testing to prioritise mismatch repair germline testing in an Australian population-based study of early onset (< 45 years old) CRC.1 In 131 patients, unselected for family history, they found 18 with germline mutations in one of the mismatch repair genes (indicating HNPCC). Based on family history alone (the Amsterdam criteria), only half of these would have been identified, indicating that tumour testing (specifically immunohistochemical staining) is a very useful adjunct to diagnosis, and more sensitive than family history. Ward et al, also Australian, have since published similar findings.2 Immunohistochemical staining has an advantage over microsatellite instability testing in that it is relatively inexpensive, can be conducted in a routine pathology laboratory (rather than a molecular laboratory), and the immunohistochemical staining test identifies the specific mismatch repair gene in which to search for a germline mutation, thereby saving money by directing the mutation search. However, although immunohistochemical staining is promising as a tool to increase diagnostic accuracy for HNPCC, it may be too early to apply this test routinely at diagnosis to all patients with CRC. There are still some problems with tumour testing. Some patients have loss of staining on immunohistochemical staining without a demonstrable mismatch repair germline mutation, indicating a lack of specificity, although this may change as germline testing improves. Immunohistochemical staining utilises antibodies that are not commonly used in pathology laboratories, and staining may be patchy and difficult to interpret for those who do not use these antibodies routinely. Moreover, there is generally a lack of understanding concerning the interpretation of loss of stain — loss of hMLH1 in a patient with an older onset CRC, without family history, usually indicates a sporadic cancer rather than HNPCC, but some of these families are now inappropriately being referred to family cancer clinics as “possible HNPCC”. Finally, some argue that tumour testing is a surrogate genetic test and so requires genetic counselling and fully informed consent. However, tumour testing should be simply viewed as a useful adjunct to HNPCC diagnosis, in that an abnormal finding on immunohistochemical staining raises the possibility, rather than the certainty, of a familial predisposition to CRC. Where to from here?Tumour testing could now be used as a “triage” measure to assist referral to a family cancer clinic for further assessment and germline testing, if appropriate. It is especially applicable when the family history approaches, but does not quite meet, the Amsterdam criteria for suspecting HNPCC or when CRC is diagnosed at an early age (younger than 50 years). However, before this can become routine, immunohistochemical staining testing for HNPCC may need to be standardised, and laboratories validated through the Royal College of Pathologists of Australia to ensure reliable results. Even so, for now, the best diagnostic precision is still achieved by a combined analysis of all the variables: family history, and clinical and pathological findings, as well as results of genetic tests.

Judy A Kirk MB BS, FRACP

One in 25 Australians carries a cystic fibrosis gene mutation, but most do not know it until they have an affected child The technology for safe, cheap screening for the principal gene mutations responsible for cystic fibrosis has long been available. Nearly 10 years ago, the US National Institutes of Health recommended “testing for gene mutations that cause [cystic fibrosis] be offered as an option to all pregnant couples and those planning a pregnancy”.1 A similar recommendation has been made by a joint committee of the American College of Obstetricians and Gynaecologists and the American College of Medical Genetics.2 In 1998, this Journal published an editorial emphasising the need for Australia to follow suit in promoting carrier screening for cystic fibrosis.3 There has been little response despite the fact that each year 70 babies are born in Australia with cystic fibrosis, almost all to parents with no family history.4 Cystic fibrosis is the most common severe autosomal recessive disease of childhood, with an incidence of 1 in 2500 and carrier frequency of 1 in 25. Clinical manifestations include progressive, irreversible suppurative lung disease and pancreatic exocrine insufficiency. Although most children with cystic fibrosis can expect to survive into adulthood, the daily therapies are rigorous, and there are many years of ill health. The median life expectancy is in the mid-30s, with end-stage lung disease the major cause of death. There is still no cure. Most patients with cystic fibrosis are detected by newborn screening, using a biochemical test (for immunoreactive trypsinogen [IRT]) for all babies, followed by cystic fibrosis gene mutation analysis for those with a raised IRT level. Newborn screening facilitates the early diagnosis of cystic fibrosis and genetic counselling for affected families. Couples identified with an affected infant can choose prenatal testing using gene mutation analysis from a chorionic villus sample for subsequent pregnancies to ascertain the status of the fetus. The genetic test used for diagnosis of cystic fibrosis and prenatal testing can also be used to identify carriers of a cystic fibrosis gene mutation. Testing for cystic fibrosis gene mutations is reliable, and, with a 12-mutation panel, nearly 85% of possible severe mutations can be detected. It can be performed using a painless cheek swab. However, testing for carrier status is generally not offered in Australia to couples without a family history, and most of those who carry a mutation do not know until an affected child is born. While we acknowledge the benefits of newborn screening, we believe it would be better to offer cystic fibrosis gene mutation screening to all couples, before they had their first baby with cystic fibrosis. Prenatal screening for a variety of conditions is routine in Australia. All women have a full blood count early in pregnancy, and those who are iron replete with a low mean cell volume are tested for thalassaemia carrier status. The outcome is that it is now uncommon for babies to be born with thalassaemia. Prenatal screening for Down syndrome (using a combination of ultrasound examination and measurement of serum markers) has been offered since 1996, and more than two-thirds of pregnant women in Victoria participate in this screening. Populations with a high frequency of genetic conditions such as Tay–Sachs disease are also offered prenatal or preconceptual screening. Paradoxically, the carrier frequency of cystic fibrosis in the general Australian population is almost the same as the carrier frequency of Tay–Sachs disease in the Ashkenazi Jewish population.5 Lack of awareness about cystic fibrosis no doubt contributes to the lack of community pressure to screen. A successful prenatal screening program for cystic fibrosis has been pursued in Edinburgh for many years.6 This program uses a model of couple testing for carrier status with the offer of prenatal genetic testing of the fetus when both partners are carriers and has halved the incidence of cystic fibrosis in that community.7 Uptake of the service is 80%,6 similar to the uptake in a smaller Dutch study.8 In Victoria, 67% of couples with an infant with cystic fibrosis have used prenatal testing for subsequent pregnancies.9 Some families have opted for pre-implantation genetic diagnosis (with in-vitro fertilisation technology) to avoid pregnancy termination. We advocate a cystic fibrosis screening program in which both parents are encouraged to be screened at the same time, which will give the most accurate risk assessment of the couple having a baby with cystic fibrosis (Box). Ideally, screening would be performed before conception, to allow the couple time to decide on the best reproductive option. In reality, many women do not present for pre-pregnancy assessment, so a screening model that allows prenatal testing of the couple should remain available. Furthermore, we advocate that both parents receive their individual carrier result to maximise the opportunities for cascade family testing. Any program offering carrier screening needs to include genetic counselling for carrier couples, individual carriers and relatives of carriers who may also wish to be tested. Extensive data clearly demonstrate the cost effectiveness of cystic fibrosis screening. The lifetime cost of care for a patient with the condition outweighs the cost of screening women of child-bearing age.10,11 Cystic fibrosis carrier screening should be a federal initiative. Currently, the care of patients with cystic fibrosis and the newborn screening programs are state funded, and there is little incentive for a national program. A Medicare-rebatable test would allow universal access and encourage uptake. Surely, it is time to fund carrier screening for cystic fibrosis in Australia. Screening for cystic fibrosis gene mutations

R John Massie PhD, FRACP · Martin B Delatycki FRACP, PhD · Agnes Bankier FRACP

The discovery of novel mutations in genes encoding succinate dehydrogenase subunits has revealed that familial phaeochromocytomas are much more common than previously thought. Genetic screening should be offered to patients with apparently sporadic phaeochromocytomas and their first-degree relatives. An increasing proportion of phaeochromocytomas present preclinically on genetic testing or as “incidentalomas” on abdominal imaging, rather than with classic symptoms and signs. Clinical suspicion should prompt measurement of plasma levels of free metanephrine or 24-hour urinary catecholamine and metanephrine levels, followed, if positive, by tumour localisation studies. With appropriate perioperative care, surgical management of phaeochromocytomas is safe and effective. Most tumours can be removed laparoscopically.

Yaser Alderazi MB BS · Michael W Yeh MD · Bruce G Robinson MD, FRACP · Diana E Benn PhD · Mark S Sywak FRACS · Diana L Learoyd PhD, FRACP · Leigh W Delbridge MD, FRACS · Stan B Sidhu PhD, FRACS

We still have no national program for detecting this potentially lethal disorder In 1985, Brown and Goldstein were awarded the Nobel Prize in Physiology and Medicine for unravelling the regulation of cholesterol metabolism in man. A key feature of their work was the elucidation of the molecular mechanism for autosomal dominant familial hypercholesterolaemia (FH), a potentially lethal disorder caused by defective endocytosis of low-density lipoprotein (LDL) cholesterol by its receptor (LDLR).1 This, in turn, led to the development of “statin” drugs, which potently lower plasma LDL cholesterol and reduce coronary heart disease (CHD) mortality. But, 20 years later, what have we achieved in detecting and treating FH? FH is characterised by lifelong marked hypercholesterolaemia (LDL cholesterol > 5 mmol/L) that leads to tissue cholesterol deposition — in such forms as tendinous xanthomata (particularly involving the Achilles), corneal arcus and palpebral xanthomas — and greatly increased risk of fatal CHD.2 Unfortunately, most people with FH are at present undiagnosed or only diagnosed after their first coronary event. We estimate that, of the roughly 40 000 cases of FH in Australia, about 20% are diagnosed and less than 10% are being adequately treated. Atherosclerosis in FH begins in early childhood. Children with FH are known to have endothelial dysfunction (the earliest phase of atherosclerosis) and increased carotid intima media thickness (CIMT), both surrogate markers of cardiovascular disease.3,4 Carotid atherosclerosis in FH rapidly progresses during childhood, at a rate proportional to plasma LDL cholesterol levels.4 FH typically involves mutations in the LDLR gene, with homozygotes having a more severe phenotype that heterozygotes. To date, about 1000 mutations have been identified in the LDLR gene (www.ucl.ac.uk/fh), most being unique, which makes the search for an unknown mutation challenging and expensive. Although heterozygous FH affects about 1 in 500 people overall,2 it occurs much more frequently in some populations such as Afrikaners, Christian Lebanese and French Canadians2 because of “founder” effects that occur when a few members of a population migrate and start a new colony. FH can be caused by mutations in genes other than LDLR. A mutation in the apolipoprotein B gene (APOB) may result in a clinical and biochemical picture that is indistinguishable from classic FH, although cholesterol levels are generally not as elevated and tendon xanthomas are less common.5 An autosomal recessive form of FH has also been described.6 The clinical picture of this condition is similar to that of homozygous FH, although it is generally less severe and more variable, with greater responsiveness to therapy. Except in “founder” populations, homozygosity for any of these conditions is exceedingly rare (about 1/1 000 000 people), and, without special intervention, such as LDL aphaeresis and liver transplantation, is typically lethal at an early age. Early statin treatment in children with FH improves endothelial function.3 A recent 2-year randomised controlled trial of pravastatin treatment (40 mg daily) in 214 children aged 8–18 years with FH showed regression of carotid atherosclerosis with no adverse effects on growth, sexual maturation, hormone concentrations, or serum liver and muscle enzyme levels.7 Despite this, the long-term safety and efficacy of statin use in children with FH is yet to be established. The Atorvastatin versus Simvastatin on Atherosclerosis Progression trial compared the effect of “aggressive” lipid-lowering treatment in FH with “conventional” lipid-lowering therapy.8 Over 2 years, LDL cholesterol lowering by high-dose atorvastatin resulted in regression of CIMT, whereas reduction with conventional-dose simvastatin did not. Moreover, the change in CIMT was proportional to the reduction in LDL cholesterol. These results support the concept that intensive lowering of LDL cholesterol levels in patients with CHD is beneficial. Although heterozygous FH patients are responsive to statins, additional treatment in combination with statins (for example, statin plus cholestyramine) is often required to achieve the desired LDL-cholesterol-lowering target.9 Moreover, combination therapy often permits use of a lower statin dose, which can benefit patients in whom adverse effects have occurred. Ezetimibe, a new drug that specifically inhibits intestinal cholesterol absorption alone, can reduce plasma LDL cholesterol concentrations by about 18%. Used in combination with a statin, it can achieve a further 25% reduction in LDL cholesterol levels over statin alone, by reducing both cholesterol supply to the liver and cholesterol biosynthesis.10 The long-term effects of ezetimibe on FH cardiovascular morbidity and mortality are unknown. The most cost-effective strategy for finding subjects with FH is to screen close relatives of patients already diagnosed with FH. Screening involves measurement of plasma LDL cholesterol, combined with either a clinical examination and family history or molecular genetic testing.11 Children born to an affected parent have a one in two risk of inheriting FH, and should be screened, at least biochemically, after the age of 2–3 years, when a cholesterol-lowering diet can be safely implemented.12 It is important to appreciate that a normal lipid profile does not rule out heterozygosity for an FH-causing mutation, particularly in early childhood.13 International experience shows that a family screening program must incorporate ethically acceptable protocols for approaching and interacting with relatives, follow-up communication with family members and their health care practitioners, as well as access to genetic counselling services, if required. Despite all these advances, it remains a tragedy that after 20 years of burgeoning knowledge about FH and the parallel development of powerful cholesterol-lowering drugs, Australia does not have a national program for detecting the vast majority of patients with FH in our community, let alone diminishing their risk of CHD.

John R Burnett MD, PhD, FRCPA · David Ravine DM, FRACP, FRCPA · Frank M van Bockxmeer BSc(Hons), PhD · Gerald F Watts DSc, MD, FRACP

Edwin P Kirk,* Annette Hattam,† Anne Turner‡ * Co-ordinator of Advanced Training, † Chair, Specialist Advisory Committee in Clinical Genetics, Royal Australasian College of Physicians; and Geneticists, Department of Medical Genetics, Sydney Children’s Hospital, High Street, Randwick, NSW 2031; ‡ Chairperson, Board of Censors in Genetic Counselling, Human Genetics Society of Australasia. kirkedATsesahs.nsw.gov.au To the Editor: Genetic risk estimation is a key element of the practice of clinical geneticists and genetic counsellors. Given this, it was with some concern that we read the findings of Bonke and colleagues regarding the performance of (mainly European) geneticists and counsellors in the application of Bayesian analysis to risk estimation.1 Bayesian analysis is taught as part of Australasian training in both clinical genetics and genetic counselling, and has been for as long as there have been formal programs. Thus, most Australian geneticists and counsellors should be familiar with the application of Bayes’ theorem to risk estimation. In actual clinical practice, it is rare to need to perform this type of analysis. This is partly because of the rapid progress in molecular genetic testing, which often obviates the need for such calculations, and partly because situations in which Bayesian analysis is clinically helpful are uncommon. Pedigrees like those in the study by Bonke et al do not come along often; when they do, the modification of prior risk by Bayesian analysis is not often important. For example, modification of a risk from 50% to 33% or from 25% to 17% (as in two of the examples used by Bonke et al) is unlikely to alter decision-making for the families involved. Specifically, as these examples all involve testing for Huntington’s disease, in which molecular analysis is usually quite straightforward, we would expect very few individuals would decide whether to proceed with testing based on being given information about modification of risk expressed this way. Moreover, when you are not performing this type of calculation regularly, it is time-consuming to do. It seems possible that many of those who completed the questionnaire would have taken greater care, and achieved greater accuracy, if faced by a real clinical situation. Nonetheless, for those of us who are involved in training clinical geneticists and genetic counsellors, the article is a useful reminder of the importance of this skill, and we will communicate with supervisors to reinforce the importance of teaching Bayesian analysis to our trainees.

Edwin P Kirk · Annette Hattam · Anne Turner