Improvement in sensitivity of allele-specific PCR facilitates reliable noninvasive prenatal detection of cystic fibrosis

BACKGROUND: Cell-free fetal DNA circulating in maternal blood has potential as a safer alternative to invasive methods of prenatal testing for paternally inherited genetic alterations, such as cystic fibrosis (CF) mutations. METHODS: We used allele-specific PCR to detect mutated CF D1152H DNA in the presence of an excess of the corresponding wild-type sequence. Pfx buffer (Invitrogen) containing replication accessory proteins and Taq polymerase with no proofreading activity was combined with TaqMaster PCR Enhancer (Eppendorf) to suppress nonspecific amplification of the wild-type allele. The procedure was tested on DNA isolated from plasma drawn from 11 pregnant women (gestational age, 11-19.2 weeks), with mutation confirmation by chorionic villus sampling. RESULTS: The method detected 5 copies of the CF D1152H mutant allele in the presence of up to approximately 100,000 copies of wild-type allele without interference from the wild-type sequence. The D1152H mutation was correctly identified in one positive sample; the only false-positive result was seen in a mishandled sample. CONCLUSIONS: This procedure allows for reliable detection of the paternally inherited D1152H mutation and has potential application for detection of other mutations, which may help reduce the need for invasive testing.     

Pharmacological mydriasis secondary to ipratropium bromide: a cause of unilateral dilated pupil.

Ipratropium bromide is a commonly used bronchodilator which has been rarely reported to cause pharmacological mydriasis. We report a 22-year-old woman using 6-hourly nebulised ipratropium from a multi-dose preparation, who presented with a unilateral dilated pupil which resolved spontaneously over 24h after discontinuation of her ill-fitting nebuliser mask which had been directing the drug toward that eye. No alternative cause was found despite extensive investigations. Greater recognition of this iatrogenic cause of reversible pupillary dilatation may avoid the need for unnecessary investigations.     

Congenital rubella cataract: a timely reminder in the new millennium?

Maternal infection with rubella in the first trimester is an important cause of congenital cataract. Any injury affecting the foetus following maternal rubella infection in the phase of organogenesis results in congenital defects collectively termed as congenital rubella syndrome (CRS). Although rubella embryopathy is a less common cause for congenital cataract than in the past, it is still seen. The number of cases reduced to one in 1997 after which there were no new cases till 2002. However, there have been two new cases of CRS in 2003. Herein another one in early 2004 is reported. Outbreaks of CRS will continue until the percentage of susceptible individuals is reduced to a minimum through immunization. The majority of rubella cases in Australia are confined to young female immigrants, many coming for marriage. We must continue to immunize children, identify and immunize vaccine failures and susceptible women before they become pregnant, and to screen pregnant women so they can be vaccinated after delivery.     

A newborn with unilateral limb enlargement

On routine neonatal examination, a newborn term male was noted to have unilateral enlargement of the right lower limb, loose thickened red skin over the palm and widening of all the fingers on the right hand. His body was pinker and warmer on the right side compared with the left and he had a right undescended testicle and hypoplastic scrotum. Radiological examination of the lower limbs demonstrated the enlargement of the soft tissue of the right lower limb compared to the left (Fig. 1). Therefore, the diagnosis was unclear from this constellation of findings and an ophthalmic assessment was requested.     

Methylenetetrahydrofolate (MTHFR), the One-Carbon Cycle,and Cardiovascular Risks

The 5-10-methylenetetrahydrofolate reductase (MTHFR) enzyme is vital for cellular homeostasis due to its key functions in the one-carbon cycle, which include methionine and folate metabolism and protein, DNA, and RNA synthesis. The enzyme is responsible for maintaining methionine and homocysteine (Hcy) balance to prevent cellular dysfunction. Polymorphisms in the MTHFR gene, especially C677T, have been associated with various diseases, including cardiovascular diseases (CVDs), cancer, inflammatory conditions, diabetes, and vascular disorders. The C677T MTHFR polymorphism is thought to be the most common cause of elevated Hcy levels, which is considered an independent risk factor for CVD. This polymorphism results in an amino acid change from alanine to valine, which prevents optimal functioning of the enzyme at temperatures above 37 °C. Many studies have been conducted to determine whether there is an association between the C677T polymorphism and increased risk for CVD. There is much evidence in favour of this association, while several studies have concluded that the polymorphism cannot be used to predict CVD development or progression. This review discusses current research regarding the C677T polymorphism and its relationship with CVD, inflammation, diabetes, and epigenetic regulation and compares the evidence provided for and against the association with CVD.     

Ultraviolet fluorescence photography: patterns in established pterygia

PURPOSE: To investigate established pterygia using our newly developed ultraviolet fluorescence photography (UVFP) system. DESIGN: Prospective observational case series. METHODS: setting: Prince of Wales Hospital, Sydney, Australia. study population: Fourteen patients (both eyes) attending the Ophthalmology Clinic at Prince of Wales Hospital for assessment of their established pterygia. There were eight men and six women, with an age range of 26 to 62 years. A total of 15 (75%) of 20 had primary pterygia, and five (25%) of 20 had recurrent pterygia. There were no specific exclusion criteria. observation procedures: Ultraviolet and standard (control) photographs were taken of the nasal and temporal interpalpebral regions bilaterally. main outcome measures: The presence of established pterygia detected by standard photography and the corresponding presence and patterns of areas of fluorescence detected by UVFP. RESULTS: In the 14 patients, 20 established pterygia were identified on standard photography. On UVFP, four patterns of fluorescence of established pterygia were identified. Of the 20 pterygia, six (30%) of 20 demonstrated fluorescence at the leading edge of the pterygium, seven (35%) of 20 demonstrated fluorescence at the limbus, three (15%) of 20 demonstrated fluorescence at both the leading edge and the limbus, and four (20%) of 20 demonstrated no visible fluorescence. CONCLUSIONS: In this study, we describe patterns of fluorescence in established pterygia by UVFP. We hypothesize that the areas of fluorescence represent areas of cellular activity within the pterygium. The patterns of fluorescence may be useful to further understand of pterygium growth and pathogenesis.     

Vitamin D,Vitamin D-Binding Proteins,and VDR Polymorphisms in Individuals with Hyperglycaemia

Vitamin D reportedly plays an important role in the pathogenesis of diabetes mellitus; however, this role is unclear and debated. This study investigated the association between 25(OH) vitamin D, vitamin D-binding proteins, and vitamin D receptor (VDR) polymorphisms in healthy individuals and those with prediabetes and type 2 diabetes mellitus (T2D) from South Africa. A cross-sectional study was conducted involving subjects of mixed ancestry aged ≥20 years. Males presented with higher mean 25(OH) vitamin D levels than females, while females exhibited significantly higher serum vitamin D-binding protein levels. Significant differences in mean 25(OH) vitamin D levels were observed in normo-glycaemic, prediabetes, screen-detected DM, and known DM individuals. Vitamin D receptor SNPs Fok1 and Taq1 were not associated with glycaemic status. Fok1 was not associated with 25(OH) vitamin D deficiency, while Taq1 was associated with vitamin D insufficiency. This study showed a high prevalence of vitamin D deficiency/insufficiency in this South African population, with decreased vitamin D levels observed in hyperglycaemic individuals, which was not linked to either vitamin D-binding protein or polymorphisms in Fok1 of the VDR gene. These results may be used as a platform for further research into diagnosis and treatment of hyperglycaemia.     

Red eye and a rodent ulcer

A 58-year-old woman presented with a 1-week history of poor vision in the right eye associated with a red eye. She was transferred for vitreoretinal management of suspected endophthalmitis. On examination, she had extensive ulceration of her forehead, exposing a pulsating mass covered with white antiseptic powder. The ulcer extended to involve the nose and right upper eyelid. Computed tomography demonstrated widespread erosion of the frontal bone, exposing the dura; histopathology confirmed the lesion to be basal cell carcinoma. The ulcer had slowly progressed for 30 years, but the patient was in denial about her condition. She had kept the ulcer well hidden by combing her hair forward over it. She was married, with an adult child, and was employed as a courier. Despite three assessments of her eye by medical staff in the preceding 24 hours, the diagnosis of the extensive basal cell carcinoma had been missed as she had refused to lift her hair for a complete examination.