In Memory of Professor Derek Pheby.

Abstract: Professor Derek Pheby's passing in November 2022 marked a profound loss for the scientific community. Professor Derek Pheby, a stalwart figure in the fields of autoimmune diseases and bioethics, was known for his dedication to scientific research and patients' support, particularly for those affected by paraneoplastic autoimmune syndromes. Professor Pheby made significant contributions to research, especially about Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). His leadership of the ME Biobank and scientific coordination of EUROMENE demonstrated his commitment to pushing boundaries and fostering international collaborations. Professor Pheby's scientific work addressed various aspects of ME/CFS, from physician education to patient needs, the development of a post-mortem tissue bank, and effective treatments. Beyond his medical career, Professor Pheby was a crucial member of the Independent Ethics Committee of MAGI, he was a poet, humanitarian, and advocate for child protection. His generosity and boundless spirit left an enduring legacy, fostering innovative research in the pursuit of combating autoimmune diseases.

Rare PECAM1 variants in three families with lymphedema.

PECAM1 is a member of the immunoglobulin superfamily and is expressed in monocytes, neutrophils, macrophages and other types of immune cells as well as in endothelial cells. PECAM1 function is crucial for the development and maturation of B lymphocytes. The aim of this study was to link rare PECAM1 variants found in lymphedema patients with the development of lymphatic system malformations. Using NGS, we previously tested 246 Italian lymphedema patients for variants in 29 lymphedema-associated genes and obtained 235 negative results. We then tested these patients for variants in the PECAM1 gene. We found three probands with rare variants in PECAM1. All variants were heterozygous missense variants. In Family 1, the unaffected mother and brother of the proband were found to carry the same variant as the proband. Lymphoscintigraphy was performed to determine possible lymphatic malformations and showed that in both cases a bilateral slight reduction in the speed and lymphatic clearance of the lower limbs. PECAM1 function is important for lymphatic vasculature formation. We found variants in PECAM1 that may be associated with susceptibility to lymphedema.

The Effects of O6-methyl Guanine DNA-methyl Transferase Promotor Methylation and CpG1, CpG2, CpG3 and CpG4 Methylation on Treatment Response and their Prognostic Significance in Patients with Glioblastoma.

This retrospective study examined the prognostic significance and treatment effect of promoter methylation of O6- methyl guanine methyl transferase (MGMT) and meth-ylation of CpG 1, CpG2, CpG3 and CpG4 in glioblastoma (GB) patients received postoperative radiotherapy (PORT), with or without adjuvant temozolomide (TMZ). One hundred patients with GB who received PORT with concomitant TMZ plus adjuvant TMZ or PORT alone, were included. The MGMT promoter methylation of CpG1, CpG2, CpG3 and CpG4 islands were examined. Overall, MGMT-methylation emerged as a significant prognostic factor for better overall survival (OS) and progression-free survival (PFS) [odds ratio (OR): 0.609, 95% confidence interval (95% CI): 0.395-0.939, p = 0.02; OR: 0.662,95% CI: 0.430-1019, p = 0.5, respectively]. The methylation of each CpG1, CpG2, CpG3 and CpG4 islands was found to have no significant effects on OS and the methylation of each CpGl, CpG2 and CpG4 islands had no significant effect on PFS (p <0.05 for all). On the other hand, the methylation of CpG3 had a positive prognostic effect on PFS (OR: 2.1, 95% CI: 0.99-4.67, p = 0.04). In the group that only received radiotherapy (RT), CpG1 and CpC3 methylations were found to have a positive prognostic significance in terms of PFS (OR: 266, 95% CI: 1.05-6.75, p -0.03 for CpG1; OR: 2.4, 95% CI: 1.01-5.92, p = 0.04 for CpG3). The MGMT promoter methylation represents an important biomarker for predicting response to therapy. Individual islands, particularly CpG3, deserves further investigation as a prognostic marker. Further studies need to be done with larger sample sizes to clarify the results.

The role of androgen receptor CAG repeat polymorphism in androgen excess disorder and idiopathic hirsutism.

PURPOSE: The study aimed to investigate whether repeat number in the androgen receptor (AR) gene has any contribution to phenotypes of the disease of androgen excess (polycystic ovary syndrome (PCOS), idiopathic hyperandrogenemia (IHA) and idiopathic hirsutism (IH) in a cohort of Turkish women. METHODS: Three hundred and fifty-four voluntary premenopausal women (172 healthy controls and 182 patients with androgen excess disorders and idiopathic hirsutism) 18-45 years of age seen at an outpatient endocrine clinic at Erciyes University Hospital between January 2013 and December 2014 were included. All volunteers have undergone physical examination and biochemical evaluation. The polymorphic (CAG)n repeat of the human AR was determined by fragment analyses. RESULTS: Detailed clinical analyses of the patients ended up with 137 PCOS, 24 IHA, and 21 IH. Pairwise comparisons revealed the CAG repeat number differences between the PCOS and controls (p = 0.005) and IH and controls (p = 0.020). Women with CAG repeat length ≤ 17 had a significantly increased twofold risk for PCOS than those women with > 17 CAG repeats OR: 2.0 (95% CI 1.2-3.3, p = 0.005). Women with CAG repeat length ≤ 17 had a significantly increased threefold risk for IH than those women with > 17 CAG repeats OR: 2.9 (95% CI 1.2-7.3, p = 0.020). When correlation analysis was performed, a weak negative correlation was detected between the short allele and FGS score (r = - 0.131, p = 0.013) and a positive relationship between total testosterone and longer allele in the IHA group (r = 0.425, p = 0.039). Median repeat length of the shorter allele between oligomenorrhea and woman with normal menstrual cycle was found to be statistically significant (p = 0.017). CONCLUSION: This study indicated that the risk of PCOS and IH is associated with the inheritance of ARs with shorter CAG repeats.

Comprehensive genotyping of Turkish women with hirsutism.

INTRODUCTION: Hirsutism is a medical sign rather than a disease affects 5-8% of women of reproductive age. Hirsutism is associated with hyperandrogenemia in most patients excluding those with idiopathic hirsutism (IH). The most common cause of hirsutism is polycystic ovary syndrome (PCOS) followed by IH and idiopathic hyperandrogenemia (IHA); however, the clinical presentation of non-classical congenital adrenal hyperplasia (NCAH) in females is often indistinguishable from other hyperandrogenic disorders with common clinical signs such as hirsutism. OBJECTIVE: The primary aim of the study is to examine the physical properties of the three genes and to make a detailed comparison of the mutations with the clinical data to contribute the etiology of hirsutism. SUBJECTS AND METHODS: 122 women admitted to the Endocrinology Clinic at Erciyes University Hospital with hirsutism were enrolled in the study between 2013-2014. All the participants were clinically evaluated. Protein-encoding exons, exon-intron boundaries of CYP21A2 (including proximal promoter), CYP11B1 and HSD3B2 genes were analyzed via state-of-the-art genetic studies. RESULTS: DNA sequencing analyses revealed two homozygous and three compound heterozygous 21-hydroxylase deficient (21OHD) NCAH patients. Additionally, three novel CYP21A2 mutations (A89V, M187I and G491S) and two novel CYP11B1 mutations (V188I and G87A) were determined. The frequencies of heterozygous mutations in CYP21A2 (including promoter), CYP11B1 and HSD3B2 genes were determined as 26.5% (15% coding region, 11.5% promoter), 11.5% and 0%, respectively. CONCLUSION: 21OHD-NCAH prevalence was determined to be ~4%. Unexpectedly, high heterozygous mutation rates were observed in CYP11B1 gene and CYP21A2 promoter region. CYP11B1 and HSD3B2 deficiencies were not prevalent in Turkish women with hirsutism despite the existence of higher heterozygous mutation rate in CYP11B1.

Diagnosis of intracranial calcification and hemorrhage in pediatric patients: Comparison of quantitative susceptibility mapping and phase images of susceptibility-weighted imaging.

PURPOSE: To prospectively compare the diagnostic capabilities of quantitative susceptibility mapping (QSM) with those of phase images of susceptibility-weighted imaging (SWI) in the detection and differentiation of intracranial calcification and hemorrhage in pediatric patients. METHOD: Sixteen pediatric patients (9 girls, 7 boys) with a mean age of 9.4±6.3 (SD) years (range, 6 days-15 years) were included. Fifty-nine calcifications and 31 hemorrhages were detected. Sensitivities and specificities of the two magnetic resonance (MR) imaging techniques were calculated and compared using McNemar test. RESULTS: QSM had a sensitivity of 84.7% and specificity of 100% for the detection of calcification. SWI phase images had a sensitivity of 49.1% and specificity of 100%. For the detection of hemorrhage, QSM had a sensitivity of 90.3% and a specificity of 98.3% whereas SWI phase images yielded a sensitivity of 64.5% and specificity of 96.6%. Overall, QSM displayed significantly better sensitivity than SWI phase images in identification of calcification and hemorrhage (P<0.05). CONCLUSION: QSM is more reliable than SWI phase images in the identification of intracranial calcification and hemorrhage in pediatric patients using MR imaging.

Genetic tests for low- and middle-income countries: a literature review.

The aim of this review is to describe a series of ten genetic diseases with Mendelian inheritance pattern in people of low- or middle-income countries, which can be easily identified with simple and affordable methods. Recent information shows that although genetic diseases account for more than 10% of infant mortality in such countries, testing, counseling, and treatment of genetic diseases is not a priority. The selection criteria for the genetic tests that are discussed in this review are: i) the frequency of the genetic disease in the general population, ii) the cost and ease of execution, and iii) the report of validated methods in the literature for the diagnosis of these diseases. The goal is to promote diagnosis of genetic diseases at low-cost and with relative ease, thereby enabling appropriate treatments, reducing mortality, and preventing genetic diseases in high-risk families.

GENETIC DISORDERS OF PITUITARY DEVELOPMENT IN PATIENTS WITH SHEEHAN'S SYNDROME.

INTRODUCTION: Genetic disorders associated with the development of the pituitary gland and cranial bones may cause a genetic tendency toward Sheehan's syndrome (SS). Our aim in this study was to investigate expression disorders in the genes responsible for the development of the pituitary gland and cranial bones in patients with SS. MATERIALS AND METHODS: Forty-four patients who were previously diagnosed with SS and 43 healthy women were compared in terms of the mean expression values of genes including the prophet of PIT-1 (PROP1), HESX homeobox 1 (HESX1), POU class 1 homeobox 1 (POU1F1), LIM homeobox 3 (LHX3), LHX4, glioma-associated oncogene homolog 2 (GLI2), orthodenticle homeobox 2 (OTX2), SIX homeobox 3 (SIX3), SIX6, T-box transcription factor 19 (TBX19), transducin-like enhancer protein 1 (TLE1), TLE3, distal-less homeobox 2 (DLX2), DLX5, MSH homeobox 2 (MSX2), and paired box 3 (PAX3). RESULTS: The mean expression values of the HESX1, TLE1, TLE3, and MSX2 genes were significantly different in the SS group from the healthy control group, while the mean expression values of the remaining genes were similar. CONCLUSION: The present study concludes that abnormal expressions of HESX1, TLE1, TLE3, and MSX2 genes may cause a genetic predisposition to the development of SS.

A huge cutaneous horn of ear.

A rare case of huge cutaneous horn of the ear was presented. The lesion was totally excised over the auricular cartilage with a 5-mm margin and the defect was closed with a full-thickness skin graft taken from the preauricular area. To the best of our knowledge, there is no any report of this kind of interesting cutaneous corn of the ear.

Partial trisomy 3q in a child with sacrococcygeal teratoma and Cornelia de Lange syndrome phenotype.

Partial duplication of 3q is a rare chromosomal disorder that leads to multiple congenital abnormalities such as growth retardation, microcephaly and characteristic facial features. Although the phenotype of the patient has similarities with Cornelia de Lange Syndrome they are etiologically different. We report here a 9 months old baby boy with partial duplication of 3q and features similar with Cornelia De Lange syndrome. Conventional cytogenetic analysis revealed a derivative chromosome 21. In order to determine the origin of this chromosome region we used subtelomeric FISH technique. Based on the results of all these cytogenetic studies and the physical examinations, the diagnosis is partial 3q duplication.

Computerized classification of intraductal breast lesions using histopathological images.

In the diagnosis of preinvasive breast cancer, some of the intraductal proliferations pose a special challenge. The continuum of intraductal breast lesions includes the usual ductal hyperplasia (UDH), atypical ductal hyperplasia (ADH), and ductal carcinoma in situ (DCIS). The current standard of care is to perform percutaneous needle biopsies for diagnosis of palpable and image-detected breast abnormalities. UDH is considered benign and patients diagnosed UDH undergo routine follow-up, whereas ADH and DCIS are considered actionable and patients diagnosed with these two subtypes get additional surgical procedures. About 250,000 new cases of intraductal breast lesions are diagnosed every year. A conservative estimate would suggest that at least 50% of these patients are needlessly undergoing unnecessary surgeries. Thus, improvement in the diagnostic reproducibility and accuracy is critically important for effective clinical management of these patients. In this study, a prototype system for automatically classifying breast microscopic tissues to distinguish between UDH and actionable subtypes (ADH and DCIS) is introduced. This system automatically evaluates digitized slides of tissues for certain cytological criteria and classifies the tissues based on the quantitative features derived from the images. The system is trained using a total of 327 regions of interest (ROIs) collected across 62 patient cases and tested with a sequestered set of 149 ROIs collected across 33 patient cases. An overall accuracy of 87.9% is achieved on the entire test data. The test accuracy of 84.6% is obtained with borderline cases (26 of the 33 test cases) only, when compared against the diagnostic accuracies of nine pathologists on the same set (81.2% average), indicates that the system is highly competitive with the expert pathologists as a stand-alone diagnostic tool and has a great potential in improving diagnostic accuracy and reproducibility when used as a "second reader" in conjunction with the pathologists.

Effects of estrogen replacement therapy on vascular endothelial growth factor expression in choroidal and retinal vasculature.

PURPOSE: To investigate the effects of estrogen replacement therapy (ERT) on vascular endothelial growth factor (VEGF) expression in choroidal and retinal vasculature in experimental rat model. METHODS: Forty female Wistar rats were randomized in 4 groups in the study. Subcutaneous ERT (17beta-estradiol, 10 microg/kg/day) was administered for three months to first group without ovariectomy and to second group with ovariectomy. Rats in third group had only ovariectomy and fourth group had sham operation. At the end of the third month, all rats were sacrificed in estrous cycles determined by vaginal smear test and their right eyes were enucleated. Enucleated eyes were analyzed by immunohistochemical method for expression of VEGF. RESULTS: Only ovariectomy (group 3) in rats did not change VEGF expression than the sham operated (group 4) rats. However, there was more VEGF expression in groups receiving ERT than the group 3 and 4, but increased VEGF expression was statistically significant in group 1 in comparison to group 3, and 4 in both, choroidal and retinal vasculature. CONCLUSION: It was shown that ERT increases VEGF expression in choroidal and retinal vasculature, in particular in non-ovariectomized rats. Our findings suggest that ERT may be a risk factor for choroidal and retinal angiogenesis. Further studies are needed to evaluate the risks or benefits of exogenous estrogen supplementation for the choroidal and retinal angiogenesis in women (Fig. 2, Ref. 25).

Response to vasoconstrictor agents by detrusor smooth muscles from cisplatin-treated rats and antioxidant treatment.

The effects of melatonin and quercetin on the contractile responses of cisplatin-treated rat detrusor smooth muscle were tested. Detrusor strips obtained from four separate rat groups (control, cisplatin, melatonin+cisplatin and quercetin+cisplatin) were mounted in 25 mL organ baths containing Krebs-Henseleit solution (KHS) at 37°C, continuously gassed with 95% O2 and 5% CO2. The vasoconstriction induced by acetylcholine (ACh) and potassium chloride (KCl) were compared within the groups. Furthermore, histopathological parameters such as edema, congestion, inflammatory cells, microvascular proliferation, fibrosis, eosinophil, mast cells and epithelial damage were noted. In routine experiments ACh and KCl triggered concentration-dependent contractions. Pretreatment with cisplatin increased the sensitivity but not the maximal response to ACh and KCl. In rats treated with melatonin or quercetin before cisplatin, the EC50 values, but not the maximal response, to both agents were significantly higher than in the cisplatin-treated (CII) group. Histopathological parameters such as edema, congestion, inflammatory cells, microvascular proliferation, fibrosis, eosinophil, mast cells and epithelial damage were all higher in the cisplatin-treated group than in the controls. Melatonin pretreatment significantly decreased mast cell numbers and epithelial damage when compared to cisplatin treatment alone but these effects were not recorded with quercetin pretreatment. These results demonstrate for the first time that melatonin can attenuate urinary bladder injury produced by cisplatin treatment.

Vasoprotection by melatonin and quercetin in rats treated with cisplatin.

Cisplatin-based chemotherapy has a variety of vascular side effects. The aim of the present study was to evaluate the beneficial effect of melatonin and cisplatin on the alterations in vascular reactivity and structure of cisplatin-treated rats. Phenylephrine (PHE) and KCl-caused concentration-dependent contractions of rat aorta. Pretreatment with cisplatin increased the sensitivity but not the max response to PHE and KCl. In rats treated with melatonin or quercetin before cisplatin, the EC50 values, but not the maximal response to both agents were significantly higher than cisplatin-treated group. Compared to the control group, cisplatin-treatment significantly reduced the luminal area of the aorta. In melatonin and quercetin-treated aortas the luminal area values were significantly higher than cisplatin-treated group. The results demonstrate for the first time that melatonin and quercetin treatment may protect the aorta in cisplatin-based chemotherapy.

Multiple-instance learning algorithms for computer-aided detection.

Many computer-aided diagnosis (CAD) problems can be best modelled as a multiple-instance learning (MIL) problem with unbalanced data, i.e., the training data typically consists of a few positive bags, and a very large number of negative instances. Existing MIL algorithms are much too computationally expensive for these datasets. We describe CH, a framework for learning a Convex Hull representation of multiple instances that is significantly faster than existing MIL algorithms. Our CH framework applies to any standard hyperplane-based learning algorithm, and for some algorithms, is guaranteed to find the global optimal solution. Experimental studies on two different CAD applications further demonstrate that the proposed algorithm significantly improves diagnostic accuracy when compared to both MIL and traditional classifiers. Although not designed for standard MIL problems (which have both positive and negative bags and relatively balanced datasets), comparisons against other MIL methods on benchmark problems also indicate that the proposed method is competitive with the state-of-the-art.

Computer-aided detection (CAD) for CT colonography: a tool to address a growing need.

Colorectal cancer is the third most common cancer in both men and women. It is estimated that in 2004, nearly 147,000 cases of colon and rectal cancer will be diagnosed in the USA, and approximately 57,000 people would die from the disease; however, only 44% of the eligible population undergoes any type of colorectal cancer screening. Many reasons have been identified for non-compliance, with key ones being patient comfort, bowel preparation and cost. Virtual colonoscopy derived from computed tomography (CT) images is gaining broader acceptance as a screening method for colorectal neoplasia. Our research suggests that computer-aided detection (CAD) as a second reader has great potential in improving polyp detection. The ColonCAD prototype presented in this paper was developed and tested on cases representative of the variability and quality in true clinical practice. Results of this study with 150 patients demonstrate that: the developed algorithm generalises well: the sensitivity for polyps > or = 6 mm is on average 90%; and the median false positive rate is a manageable 3 per volume.

Changes in urethral connective tissue after estrogen replacement therapy in ovariectomized rats.

OBJECTIVE: Genuine stress urinary incontinence is a common problem after the middle age. Various treatment modalities have been tried as a treatment for this entity and estrogens are one of the most common alternatives. Their effects on the urethral mucosa have been described in many studies, while the impact on the urethral connective tissue has been evaluated only in a few reports. In this study, we aimed to investigate the effect of estrogen on urethral connective tissue in ovariectomized rats. MATERIAL AND METHODS: Thirty-six Wistar rats (4 months old, 250-300 g weight) were included in the study. They were organized as group 1 (ovariectomized, n = 10), group 2 (ovariectomized + late estrogen treatment, n = 10), group 3 (ovariectomized + early estrogen treatment, n = 10) and group 4 (sham operated, n = 6). Rats in group 1 were killed 6 weeks after the ovariectomy. Group 2 was ovariectomized 6 weeks before treatment and then received estradiol for 6 weeks. Group 3 was treated with estradiol for 6 weeks immediately after the ovariectomy. After killing the rats, urethral tissues were removed. Quantitative analysis of collagen fiber content was done by computerized image analyzer system in specimens stained with Masson's trichrome technique. RESULTS: Percentages of collagen fiber content (mean +/- standard error of mean) were 58.4 +/- 5.9% in group 1; 53.3 +/- 4.8% in group 2; 46.3 +/- 3.4% in group 3 and 40.2 +/- 3.2% in group 4. There was not any significant difference among the groups (p > 0.05). CONCLUSIONS: Our results suggest that early and late estrogen replacements do not seem to have a discriminating effect on the urethral connective tissue. Further studies are needed to assess the preventive effect of estrogen on urethra.

Pro-inflammatory cytokine response of the fluid contents of spermatoceles and epididymal cysts.

The aim of the study was to determine the cellular contents and concentrations of interleukin 6 (IL-6), interleukin 8 (IL-8) and tumour necrosis factor alpha (TNF-alpha) in fluids of patients with spermatocele or epididymal cyst. Twenty-five symptomatic patients, 14 with epididymal cysts and 11 with spermatoceles, were included in the study. Fluids were obtained during surgical excision of the cysts and cytological smears were stained with May-Gruenwald-Giemsa to establish cell components. The concentrations of IL-6, IL-8 and TNF-alpha were measured by chemiluminescent immunometric assay. Cytological analysis of the fluids demonstrated various sperm forms ranging from immature germ cells to degenerated spermatozoa without inflammatory cells such as neutrophils and macrophages. The concentrations (mean+/- SEM, pg/mg protein) of IL-6, IL-8 and TNF-alpha were 13.52 +/- 1.40, 22.20 +/- 2.43, 3.51 +/- 1.43 in spermatocele fluids and 5.76 +/- 0.48, 11.57 +/- 1.89, 2.53 +/- 0.41 in epididymal cyst fluids. Both IL-6 and IL-8 concentrations in the spermatocele group were higher than in the epididymal cyst group (P < 0.0001). There were no differences in TNF-alpha concentrations between the groups (P > 0.05). These findings indicate that local production of pro-inflammatory cytokines is involved in cyst formation. The presence of immunologic activation in these fluids advocates a policy of selective surgical intervention in patients with spermatocele or epididymal cyst.

A new experimental model for cryptorchidism: inguinoscrotal approach.

We investigated the effect of inguinal canal closure as a new mechanically induced cryptorchid rat model. The effectiveness of this new model was evaluated by histopathological examination. Thirty-one 21-day-old Wistar rats were divided into four groups. In groups 1 (n = 6), 2 (n = 6) and 3 (n = 7), unilateral undescended testis was created by performing inguinal canal closure with inguinoscrotal approach. Sham-operated rats were used as controls in group 4 (n = 12). The rats were killed on day 30 after surgery in group 1, day 45 in group 2 and day 60 in group 3. The seminiferous tubular diameter, number of tubules with mature germ cell and Leydig cell clusters were evaluated. None of the rats were lost during the study period. Signs of infection were not detected in operation site although antibiotics were not used. Overall only three (16%) testes descended into scrotum in study groups. The operation time was 3-4 min for each rat. Histopathological examination revealed detrimental effects of cryptorchidism on testicular growth in study groups. In all groups, except the sham group, the mean tubular diameter and the number of tubules with mature germ cells in the left testicle were significantly decreased compared to the right ones. Our findings were in correlation with other experimental studies using different rat models of cryptorchidism. This new model of cryptorchidism is considered to provide a simple and effective technique for investigating the impaired development of the testes in cryptorchidism.

Detection of mutations in the RB1 gene by single strand conformation polymorphism (SSCP) analysis, amplification mismatch detection (AMD) analysis and polymerase chain reaction sequencing.

Mutations of the retinoblastoma gene are known to cause both nonhereditary and hereditary forms of retinoblastoma. Most patients with hereditary retinoblastoma have bilateral disease. Hereditary predisposition to retinoblastoma is caused by a germline mutation at the retinoblastoma gene locus (RB1) and transmitted as an autosomal dominant trait with 90% penetrance. Three quarters of these alterations represent de novo mutations. Since 75% of these cases are new mutations, there is a need for methods which can be used to identify carriers, so that informed genetic counselling will be available to patients and close relatives. In the present study, leukocyte DNA and RNA from 5 patients with sporadic bilateral retinoblastoma. were subjected to single stranded conformation analysis (SSCP) and amplification and mismatch detection (AMD) analysis. SSCP band shifts were found in 3 of the 5 patients. AMD was applied to reverse-transcriptase PCR and exons of the RB1 gene in the patients with bilateral retinoblastoma. Cleavage was found in 2 patients. Neither of these patients corresponded to the 3 with SSCP band shifts. Thus in total, 5 patients with retinoblastoma had mutations detected by a combination of SSCP and AMD analysis, and proof was sought by means of sequencing. This approach has proved to be a useful method for the rapid detection of mutations in the RB1 gene. The five mutations detected in this study were all novel and emphasise the heterogeneity of the molecular pathology in this gene.