[Application of molecular genetics in microdeletion syndromes: report of the results of 21 FISH tests for Williams Beuren syndrome 3rd part of microdeletion project]

Twenty one FISH tests for confirmation of Williams Beuren Syndrome [19 Peripheral blood, and two amniotic cells cultures] have been performed. FISH tests in four couples with one affected child of Williams Syndrome are negative for abnormal critical segment of del [7p11-23], which indicates that the deletion in the affected children is of de novo origin. There are two negative FISH tests out of 8 candidates suspected for WS. One is positive for Prader-Willi Syndrome, the other one is negative for WS, so far. One negative FISH test out of 8 indicates the importance of FISH test in detection of microdeletion disease, especially in Williams Beuren Syndrome

[Application of molecular genetics in microdeletion diseases: report of the results 41 FISH tests for critical region segment of deletion 15q11-13 [Prader-Willi / Angelman syndrome]]

In spite of tiny deletion of chromosomal segment in microdeletion diseases the affected individual represents severe clinical manifestation. The classical cytogenetic techniques including high resolution are unable to clarify the deleted segment. It is worthy performing FISH study for clinically suspected patients. The result of 39 blood samples and two amniotic fluid samples studied by FISH of peripheral blood [1-24 years old] and two amniotic fluid including: peripheral blood samples clinically suspected for Angelman syndrome, 15 cases for Prader-Willi syndrome and five children PB and two amniotic fluid for PWS/ AS syndrome are as follows: Seven samples of Angelman syndrome, three cases of Prader-Willi syndrome, and 2 cases of other samples totally, 12 of 40 [30%] were positive for deletion of critical segment 15 [q11-q13], confirming the clinical diagnosis. One blood sample for Angelman syndrome did not growth

[Williams syndrome: report of a case]

Williams syndrome is one of mental retardation reasons. Most cases are sporadic but parent to child transmission has been reported. Patients have peculiar face, namely "elfin facies", with periorbital fullness, epicanthal folds, depressed nasal bridge, anteverted nares, and full lips. Cardiac malformation are supravalvular aortic stenosis, pulmonic valvular stenosis, ventricular and arterial septal defect. IQ is ranged from 40 to 80. Deletion within chromosome 7q11.23 is the reason in both sporadic and inherited cases. We are reporting a 3-year old boy, with mental retardation, periorbital fullness, full lips and cardiac malformation. In FISH studies, deletion of short arm of chromosome 7, confirmed the diagnosis of Williams syndrome

Deletions in the survival motor neuron gene in Iranian patients with spinal muscular atrophy

<p><b>INTRODUCTION</b>Spinal muscular atrophy (SMA) is a common neuromuscular disorder with progressive paralysis caused by the loss of alpha-motor neurons in the spinal cord. The survival motor neuron (SMN) protein is encoded by 2 genes, SMN1 and SMN2. The most frequent mutation is the biallelic deletion of exon 7 of the SMN1 gene. In SMA, SMN2 cannot compensate for the loss of SMN1, due to the exclusion of exon 7. The aim of our study was to estimate the frequency of the common SMN1 exon 7 deletion in patients referred to our centre for carrier detection and prenatal diagnosis.</p><p><b>MATERIALS AND METHODS</b>We performed the detection of exon 7 deletion of the SMN1 gene for the affected patients and fetuses suspected to have SMA.</p><p><b>RESULTS</b>Of 243 families, 195 were classified as SMA type I, 30 as type II, and 18 as type III according to their family histories. The analysis of exon 7 deletion among living affected children showed that 94% of the patients with SMA type I, 95% with type II families and 100% with type III had homozygous deletions. Of the prenatal diagnoses, 21 (22.8%) of the 92 fetuses were found to be affected and these pregnancies were terminated.</p><p><b>CONCLUSIONS</b>The homozygosity frequency for the deletion of SMN1 exon 7 for all 3 types was (94%), similar to those of Western Europe, China, Japan and Kuwait.</p>

[ role of FISH in prenatal diagnosis of chromosomal anomalies]

FLUORESCENT in situ hybridization has multiple applications in prenatal diagnosis. It can be used for the detection of common chromosome aneuploidies of chromosomes 13,18,21, X and Y it can be used for the early detection of segmental imbalances detected in former affected offspring or resulting from balanced translocations in the carrier parents. FISH is also a useful tool for ruling in/out of mosaicism detected in routine chromosomal study as it will provide a means of screening and scoring a larger number of cells than theoretically possible with routine chromosomal study. In this report we are presenting results of prenatal diagnosis cases where we have employed FISH techniques for direct analysis or for confirmation of results otherwise obtained. In a two year period 60 FISH tests have been performed on fetal samples. Forty five cases were tested for 5 common chromosomes ten of these cases were referred for advanced maternal age, 29 for abnormal marker screening test results, and 6 for history of former offspring with chromosome aneuploidy. The remaining cases were tested for other reasons including 4 for microdeletion syndromes previously detected in other offspring, 4 for mosaicism suspected in routine chromosomal study, and 7 for possible imbalance resulting from known chromosome translocation in either parent. In all cases, the results were confirmed with another technique, most often the results of routine chromosomal study and in microdeletion cases, reanalysis on metaphases prepared from cultured cells. There was 100% correlation between the results obtained from the two techniques. Our experience shows that this technique has the advantage of a speedy result with considerable reliability for use in the detection of specific cases. We recommend the application of FISH for all high risk pregnancies

[Craniosynostosis in a patient with 2q37.3 del 5q34dup: association of extra copy of MSX2 with craniosynostosis]

We report a 1-year-old boy with craniosynostosis, microcephaly, developmental delay and dysmorphic features. His karyotype was 46,XX,add2q. Chromosomal study of parents was normal. The imbalance on chromosome 2 in the patient was further defined using comparative genomic hybridization [CGH], which revealed a loss of material from 2q37.3-qter and a gain of 5q34-qter. Florescent in situ hybridization [FISH] studies using subtelomeric 2q and 5q were used to confirm 2q deletion and 5q duplication. It demonstrated 3 signals for 5q terminal and 1 signal for 2q terminal. FISH studies of parents were normal. Recently it has been proposed that an extra copy of the MSX2 gene that is mapped to 5q35.2 causes premature synostosis of the sutures via the MSX2-mediated pathway of calvarial osteogenic differentiation. Our case gives further support of the role of extra copy of MSX2 gene leading to craniosynostosis

[Cytogenetic analysis for Smith-Magenis syndrome in an Iranian family]

Smith-Magenis syndrome [SMS] is a multisystem, neurodevelopmental genetic disorder associated with mental retardation that predisposes individuals to a distinct pattern of maladaptive behaviors and other neuropsychological impairments. A family with a mental retarded male individual suspected of this syndrome were referred to our center for prenatal diagnosis. After genetic counseling, karyotyping was performed for confirmation of the disease. The results indicated that both parents were normal and the affected child had del[17][p11.2 [right arrow]p12]. The chromosomal anomaly with facial characteristics not particularly evident, mental retardation, behavioral troubles, hyperactivity, autistic traits and dysarticulation led to the diagnosis of the Smith-Magenis syndrome

[Aspects of Greig cephalopolysyndactyly syndrome in a patient with 7pl3pl5 deletion]

A 1-month-old infant with some aspects of Greig cephalopolysyndactyly syndrome [GCPS] and additional features is described. High-resolution chromosomal analysis showed a de novo interstitial deletion of chromosome 7p with breakpoints located at p13 and p15. Our case has some additional features of GCP syndrome, such a cleft palate, narrow auditory canal, umbilical hernia, inguinal hernia, hirsutism, sacral dimple and small skin tag above natal cleft. Frontal bossing, macrocephaly, and high forehead, features associated with GCP was absent in our patient. Hirsutism has been previously reported in only 5 patients with 5p13 deletion. Based on those cases, it has been suggested that the responsible gene might be located in that region. Our patient gives further evidence of this hypothesis

[Survival motor neuron gene deletions among Iranian patients with spinal muscular atrophy]

Spinal muscular atrophy [SMA] is a common neuromuscular disorder with progressive paralysis caused by the loss of alpha-motor neuron in the spinal cord. SMN is encoded by two genes, SMN1 and SMN2, which essentially differ by a single nucleotide in exon 7. The most frequent mutation is biallelic deletion of exon 7 of the SMN1 gene. A small percentage of SMA patients present compound heterozygosity with a point mutation on one allele and deletion on the other. In the remaining cases, the disease is unlikely to be related to SMN1 defects. In spinal muscular atrophy [SMA], SMN2 is not able to compensate for the loss of SMN1 due to exclusion of exon 7. The aim of our study was to estimate the frequency of the common exon 7 SMN1 deletion in the families who referred to our center for carrier detection and prenatal diagnosis. Between March 1999 and March 2006, one hundred sixty seven families with history of at least one affected member were referred to us. We performed detection of deletion exon 7 SMN1 for the patients and carrier detection for their parents, prenatal diagnosis in subsequent pregnancies to couples who previously had an affected child became possible [63 prenatal diagnosis]. From 167 families, 139 categorized in type I of the disease, 21 in type II, and 7 in type III. Carrier detection for the parents indicated that in 96 families with history of affected member with type I SMA both parents carried the deletion in exon 7 and in 20 families, one of the parents was carrier. These rates were 16 to 1 for SMA type II, and 3 to 2 for type III SMA. Sixty-four children affected with SMA were studied, 58 of them were found to be homozygous for the loss of exon 7 of the SMN1 gene, except two patients who were heterozygote for exon 7 deletion [frequency of homozygocity: 90.7%]. Eleven of sixty-three [17.5%] fetal samples were found to be affected and these pregnancies were terminated. The molecular analysis of the biallelic exon 7 of the SMN1 deletion is a standard and reliable test in cases of SMA

[Seventeen years experience in precise diagnosis and prenatal diagnosis of mucopolysaccharidoses [MPS]]

Upon a scientific collaboration, families having affected offspring suspected for MPS disease were enzymaticaly analyzed. In 82 families the deficit enzymes were detected. Seventy prenatal diagnosis for parous at risk were performed, revealing 53 unaffected and 17 affected fetuses. All families with affected fetuses opted for pregnancy termination. The prenatal result of unaffected newborns confirmed the prenatal diagnosis findings. The summary of clinical findings and epidemiological distribution of MPS disorders and PND results are presented in this short report

Comparison of early and mid-trimester amniocentesis in 1459 amniotic fluid cultures

Early amniocentesis is a new context in prenatal diagnosis. However, the consequences of this procedure are not known clearly. We compared cytogenetic results of 655 amniotic fluid samples obtained at 12-14 gestational weeks [early amniocentesis, EA] and 804 samples at 15-18 gestational weeks [mid-trimester amniocentesis, MA]. The rate of chromosomal abnormalities for early amniocentesis was 3.2% [21 cases] and for mid-trimester amniocentesis was 5.3% [43 cases] [p=0.047]. True mosaicism was seen in 2 cases MT group [p=1.000]. We did not have maternal cell contamination in either group. The ratio of repeat amniocentesis was 0.6 percent in the EA group compared with 0.2 percent in MA group [p=0.417]. Procedure-related early abortion [within the first 30 days after amniocentesis] was seen in 4 cases of EA [2.2%] and 3 cases of MA [1.5%] [p=0.719]. Hemorrhage occured in the same ratio. Intra-uterine fetal death [IUFD] was seen in 7 cases of MA, but not in any cases of EA [p=0.015]. Our findings showed comparable outcome in two method of amniocentesis, except for lower chromosomal abortion rate and IUFD

Overview of lipid storage diseases and 16 years experience of biochemical analysis and prenatal diagnosis in Iran

Lipid storage diseases are a group of metabolic disorders characterized by an enzyme defect leading to progressive accumulation of heterogeneous undigested lipid substance in the lysosomal organelles, causing variety of diseases according to defective gene and accumulated lipid in the different organ1 s cells.This study was based on enzymatic assays were performed for 409 affected members of two hundred and thirty-three families whom we had screened for metabolic disorders from August 1990 to November 2006. For ruling in/out of the suspected disorder, assay of urine, blood and skin fibroblasts were performed in Erasmus University. The necessary samples were taken according to the established protocols. Among the received 409 samples, 158 samples were suspected to have some forms of lipid storage diseases, 48 did not have the suspected enzyme defect and no diagnosis was established. In 84 cases, a diagnosis was reached with the first enzyme assay. In the remaining 25 cases, the first diagnosis which was metachromatic leukodystrophy in the majority of cases was negative and the second enzyme assay for another disorder after further workup proved to be positive and diagnostic. After enzyme assay 114 individuals were shown to have some lipid storage disorder.The highest number of cases studied is the Neimann-Pick cases with 22 members from 14 families, followed by 17 members from 16 families with Metachromatic leukodystrophy, 14 cases from 11 families referred for Gaucher and 10 families with 10 affected members with Tay Sachs. In our experience, emphasis on clinical workup prior to testing can cut down unnecessary expenses, time and effort

History of hemoglobinopathies and prenatal diagnosis in Iran: report of 1141 cases

Hemoglobin is the first polypeptide that showed a direct relation between the gene and its product. Hemoglobin is a tetramer composed of a couple of different globin chains surrounding the heme nucleus. In addition to different original hemoglobin, according to the point changes in the globin genes, different hemoglobin molecules may be formed, but almost similar to original hemoglobin. This may be a polymorphism phenomenon. Some of these point mutations result in abnormal hemoglobin such as HbS and HbC which causes sickle cell anemia and hemoglobin C disease. In a healthy human; there are three kinds of hemoglobin designated as HbA[1][97%] A[2] [3%] and F [0.3%] composed of alpha2 beta2, alpha2, and alpha2lambda2 respectively. Mutation on a or beta chain gene may result to abnormal hemoglobins causing mild to severe disorders. These disorders are frequent in the Mediterranean and Middle East countries. The first prenatal diagnosis [PND] for thalassemia was performed in 1972 and improving very well. The first PND for thalassemia in spite of lots of problems has been done in our country at 1990, and then later on a national prevention net has been established working very well, which could be a good model for neighbor countries. According to the different genes and their frequencies over the country; we divided the country to eight parts. Upon the gene frequencies, 23 mutations of beta globin and 21 mutations of alpha globin gene has been detected. So far, we believe that there are still some other mutation genes, as well. We are reporting the result 1141 PND of hemoglobinopathies. In our study 23.2% of fetuses were normal, 48.3% were minor thalassemia and 23.2% were affected bearing major beta thalassemia. In 2.8% of cases; we were able to detect one normal allele, therefore fetuses may be normal or minor thalassemia. In 0.9% only one mutant gene has been detected, thus fetus may be major or minor thalassemia. We were unable to identify none of genes in 0.9% cases

Highly sensitive conventional PCR testing of free fetal DNA in maternal serum for non-invasive fetal sex determination

Background: free fetal DNA [FFD] in maternal plasma/serum has increasingly become the source of fetal material for diagnostic purposes in recent years. This source of fetal material can be used for sex determination, Rh typing, paternally inherited sequences and compound heterozygosity. Reports on the lack of consistent PCR amplification of Y-chromosome sequences of FFD in maternal plasma/serum have led diagnostic services to the use of real-time PCR for improved sensitivity of sex determination. We report the use of conventional PCR for fetal sex determination with high sensitivity and reproducibility

Methods: peripheral blood samples were obtained from 21 pregnant volunteers during 4-29 weeks of gestation. A healthy man and 52 healthy non-pregnant women were investigated in this study as controls. All the samples were collected at random. Fetal gender was determined by conventional PCR to detect a Y-chromosomal sequence [DYZ1] in maternal serum and confirmed by amniocentesis or after delivery

Results: fetus-derived Y sequences were detected in 16 out of 17 maternal serum samples with male fetuses and none of the 4 women bearing female fetuses had positive results. The sensitivity of our PCR reached >95%, and the specificity reached >96% [Y signal detected in only 2 out of 52 female samples studied]

Conclusion: our report on the improved sensitivity of Y-chromosome PCR simplifies routine sex determination in diagnostic laboratories without the use of real-time PCR

Cytogenetic analysis of 358 bone marrow samples of suspected Acute Lymphoblastic Leukemia patients

Acute Lymphoblastic leukemia [ALL] is characterized by the accumulation of malignant, immature lymphoid cells in the bone marrow and in most cases also in peripheral blood. The disease is much more common in children than in older age group, with the incidence peaking around 3-5 years of age. Initial cytogenetic studies showed chromosomal aberrations in approximately half of all patients with ALL. Later studies and the combined use of FISH and molecular techniques have shown that the rates of detected chromosomal aberrations are 70% to 80% in these patients. The rates of detection in ALL have never been reported in Iran and we are reporting our cytogenetic findings in 358 ALL cases. We evaluated the karyotypes of 358 [21%] bone marrow samples sent to our center for cytogenetic study from patients with probable clinical diagnosis of ALL. All samples were submitted to a cell count and cultures were set up accordingly using standard protocols. Chromosomal aberrations were detected in 155 [49%] out of 314 successful cultures and the remaining 159 [51%] showed normal karyotypes. Hyperdiploidy in two forms of moderate and massive was the most common chromosomal aberration, found in 63 [40%] cases, followed by 14q abnormality in 13 [8%] and deletion of long arm of chromosome 6 in 9 [6%] of the cases. The ratios of detected numerical and structural chromosomal changes are close to and comparable with various series studies based on banding cytogenetic techniques alone. We believe that the implementation of complementary FISH and molecular cytogenetic techniques, and closer communication with the referring physicians are extremely instrumental in improving the effectiveness of cytogenetics study