Multiplex ligation-dependant probe amplification study of children with idiopathic mental retardation in South India

BACKGROUND:

Mental retardation (MR) is a heterogeneous dysfunction of the central nervous system exhibiting complex phenotypes and has an estimated prevalence of 1-3% in the general population. However, in about 50% of the children diagnosed with any form of intellectual disability or developmental delay the cause goes undetected contributing to idiopathic intellectual disability.

MATERIALS AND METHODS:

A total of 122 children with developmental delay/MR were studied to identify the microscopic and submicroscopic chromosome rearrangements by using the conventional cytogenetics and multiplex ligation dependent probe amplification (MLPA) analysis using SALSA MLPA kits from Microbiology Research Centre Holland [MRC] Holland.

RESULTS:

All the recruited children were selected for this study, after thorough clinical assessment and metaphases prepared were analyzed by using automated karyotyping system. None was found to have chromosomal abnormality; MLPA analysis was carried out in all subjects and identified in 11 (9%) patients.

CONCLUSION:

Karyotype analysis in combination with MLPA assays for submicroscopic micro-deletions may be recommended for children with idiopathic MR.     

Utility of MLPA in mutation analysis and carrier detection for Duchenne muscular dystrophy

CONTEXT:

Multiplex ligation probe amplification (MLPA) is a new technique to identify deletions and duplications and can evaluate all 79 exons in dystrophin gene in patients with Duchenne muscular dystrophy (DMD). Being semi-quantitative, MLPA is also effective in detecting duplications and carrier testing of females; both of which cannot be done using multiplex PCR. It has found applications in diagnostics of many genetic disorders.

AIM:

To study the utility of MLPA in diagnosis and carrier detection for DMD.

MATERIALS AND METHODS:

Mutation analysis and carrier detection was done by multiplex PCR and MLPA and the results were compared.

RESULTS AND CONCLUSIONS:

We present data showing utility of MLPA in identifying mutations in cases with DMD/BMD. In the present study using MLPA, we identified mutations in additional 5.6% cases of DMD in whom multiplex PCR was not able to detect intragenic deletions. In addition, MLPA also correctly confirmed carrier status of two obligate carriers and revealed carrier status in 6 of 8 mothers of sporadic cases.     

Chromosomal abnormalities in couples with repeated fetal loss: An Indian retrospective study

BACKGROUND:

Recurrent pregnancy loss is a common occurrence and a matter of concern for couples planning the pregnancy. Chromosomal abnormalities, mainly balanced rearrangements, are common in couples with repeated miscarriages.

PURPOSE:

The purpose of this study is to evaluate the contribution of chromosomal anomalies causing repeated spontaneous miscarriages and provide detailed characterization of a few structurally altered chromosomes.

MATERIALS AND METHODS:

A retrospective cytogenetic study was carried out on 4859 individuals having a history of recurrent miscarriages. The cases were analyzed using G-banding and fluorescence in situ hybridization wherever necessary.

RESULTS:

Chromosomal rearrangements were found in 170 individuals (3.5%). Translocations were seen in 72 (42.35%) cases. Of these, reciprocal translocations constituted 42 (24.70%) cases while Robertsonian translocations were detected in 30 (17.64%) cases. 7 (4.11%) cases were mosaic, 8 (4.70%) had small supernumerary marker chromosomes and 1 (0.6%) had an interstitial microdeletion. Nearly, 78 (1.61%) cases with heteromorphic variants were seen of which inversion of Y chromosome (57.70%) and chromosome 9 pericentromeric variants (32.05%) were predominantly involved.

CONCLUSIONS:

Chromosomal analysis is an important etiological investigation in couples with repeated miscarriages. Characterization of variants/marker chromosome enable calculation of a more precise recurrent risk in a subsequent pregnancy thereby facilitating genetic counseling and deciding further reproductive options.     

Chromosomal abnormalities as a cause of recurrent abortions in Egypt

BACKGROUND:

In 4%-8% of couples with recurrent abortion, at least one of the partners has chromosomal abnormality. Most spontaneous miscarriages which happen in the first and second trimesters are caused by chromosomal abnormalities. These chromosomal abnormalities may be either numerical or structural.

MATERIAL AND METHODS:

Cytogenetic study was done for 73 Egyptian couples who presented with recurrent abortion at Genetic Unit of Children Hospital, Mansoura University.

RESULTS:

We found that the frequency of chromosomal abnormalities was not significantly different from that reported worldwide. Chromosomal abnormalities were detected in 9 (6.1%) of 73 couples. Seven of chromosomal abnormalities were structural and two of them were numerical.

CONCLUSION:

Our results showed that 6.1% of the couples with recurrent abortion had chromosomal abnormalities, with no other abnormalities. We suggest that it is necessary to perform cytogenetic in vestigation for couples who have recurrent abortion.     

Impact of pericentric inversion of Chromosome 9 [inv (9) (p11q12)] on infertility

BACKGROUND:

One of the frequent occurrences in chromosome rearrangements is pericentric inversion of the Chromosome 9; inv (9) (p11q12), which is consider to be the variant of normal karyotype. Although it seems not to correlate with abnormal phenotypes, there have been many controversial reports indicating that it may lead to abnormal clinical conditions such as infertility. The incidence is found to be about 1.98% in the general population.

MATERIALS AND METHODS:

We investigated the karyotypes of 300 infertile couples (600 individuals) being referred to our infertility clinic using standard GTG banding for karyotype preparation.

RESULTS:

The chromosomal analysis revealed a total of 15 (2.5%) inversions, among these, 14 male patients were inversion 9 carriers (4.69%) while one female patient was affected (0.33%). The incidence of inversion 9 in male patients is significantly higher than that of normal population and even than that of female patients (P< 0.05).

CONCLUSIONS:

This result suggests that inversion 9 may often cause infertility in men due to spermatogenic disturbances, which are arisen by the loops or acentric fragments formed in meiosis.     

A modified multiplex ligation-dependent probe amplification method for the detection of 22q11.2 copy number variations in patients with congenital heart disease

Background

Copy number variations (CNVs) of chromosomal region 22q11.2 are associated with a subset of patients with congenital heart disease (CHD). Accurate and efficient detection of CNV is important for genetic analysis of CHD. The aim of the study was to introduce a novel approach named CNVplex®, a high-throughput analysis technique designed for efficient detection of chromosomal CNVs, and to explore the prevalence of sub-chromosomal imbalances in 22q11.2 loci in patients with CHD from a single institute.

Results

We developed a novel technique, CNVplex®, for high-throughput detection of sub-chromosomal copy number aberrations. Modified from the multiplex ligation-dependent probe amplification (MLPA) method, it introduced a lengthening ligation system and four universal primer sets, which simplified the synthesis of probes and significantly improved the flexibility of the experiment. We used 110 samples, which were extensively characterized with chromosomal microarray analysis and MLPA, to validate the performance of the newly developed method. Furthermore, CNVplex® was used to screen for sub-chromosomal imbalances in 22q11.2 loci in 818 CHD patients consecutively enrolled from Shanghai Children’s Medical Center. In the methodology development phase, CNVplex® detected all copy number aberrations that were previously identified with both chromosomal microarray analysis and MLPA, demonstrating 100% sensitivity and specificity. In the validation phase, 22q11.2 deletion and 22q11.2 duplication were detected in 39 and 1 of 818 patients with CHD by CNVplex®, respectively. Our data demonstrated that the frequency of 22q11.2 deletion varied among sub-groups of CHD patients. Notably, 22q11.2 deletion was more commonly observed in cases with conotruncal defect (CTD) than in cases with non-CTD (P < 0.001). With higher resolution and more probes against selected chromosomal loci, CNVplex® also identified several individuals with small CNVs and alterations in other chromosomes.

Conclusions

CNVplex® is sensitive and specific in its detection of CNVs, and it is an alternative to MLPA for batch screening of pathogenetic CNVs in known genomic loci.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1590-5) contains supplementary material, which is available to authorized users.     

Cytoprotective effect of honey against chromosomal breakage in fanconi anemia patients in vitro

BACKGROUND:

Natural honey is widely used all over the world as a complementary and alternative medicine in various disorders including Fanconi anemia (FA). FA is a rare genetic chromosomal instability syndrome caused by impairment of DNA repair and reactive oxygen species (ROS) imbalance. This disease is also related to bone marrow failure and cancer. The aim of this study was to evaluate the cytoprotective effect of honey on mitomycin C (MMC-) induced chromosomal damage in peripheral lymphocytes from FA patients.

MATERIALS AND METHODS:

Treatment of these complications with alkylation agents MMC may enhance chromosomal breakage. We have evaluated the effect of honey on MMC- induced chromosomal breakage in FA blood cells using chromosomal breakage assay. The basal chromosomal breakage count was higher among FA patients than healthy subjects.

RESULTS:

The addition of MMC alone gave a significantly higher of chromosomal breakage in FA patients than control group (P < 0.0001). Pre- treatment with honey significantly inhibited breakage induced by MMC in FA patients by its antioxidant effect.

CONCLUSION:

Honey can prevent MMC- induced chromosomal breakage by its antioxidant effect.     

Genomic characterization of some Iranian children with idiopathic mental retardation using array comparative genomic hybridization

BACKGROUND:

Mental retardation (MR) has a prevalence of 1-3% and genetic causes are present in more than 50% of patients. Chromosomal abnormalities are one of the most common genetic causes of MR and are responsible for 4-28% of mental retardation. However, the smallest loss or gain of material visible by standard cytogenetic is about 4 Mb and for smaller abnormalities, molecular cytogenetic techniques such as array comparative genomic hybridization (array CGH) should be used. It has been shown that 15-25% of idiopathic MR (IMR) has submicroscopic rearrangements detectable by array CGH. In this project, the genomic abnormalities were investigated in 32 MR patients using this technique.

MATERIALS AND METHODS:

Patients with IMR with dysmorphism were investigated in this study. Karyotype analysis, fragile X and metabolic tests were first carried out on the patients. The copy number variation was then assessed in a total of 32 patients with normal results for the mentioned tests using whole genome oligo array CGH. Multiple ligation probe amplification was carried out as a confirmation test.

RESULTS:

In total, 19% of the patients showed genomic abnormalities. This is reduced to 12.5% once the two patients with abnormal karyotypes (upon re-evaluation) are removed.

CONCLUSION:

The array CGH technique increased the detection rate of genomic imbalances in our patients by 12.5%. It is an accurate and reliable method for the determination of genomic imbalances in patients with IMR and dysmorphism.     

Cytogenetic abnormalities in 222 infertile men with azoospermia and oligospermia in Iran: Report and review

BACKGROUND:

Infertility affects approximately 10%-15% of couples in reproductive age. In half of the couples, causes are male-related, associated with impaired spermatogenesis. There is a complex correlation between genetics and infertility. Several factors affect on gametogenesis, from which factors that lead to chromosomal abnormalities are one of the best known. The aim of this study was to determine type and rate of chromosomal abnormalities in infertile azoospermic and oligospermic males in Iranian population.

MATERIALS AND METHODS:

The records of a total of 222 participants were evaluated retrospectively.

RESULTS:

As a whole we observed 13.96% chromosomal abnormality, from which 12.15% showed numerical and 1.8% showed structural abnormalities.

CONCLUSION:

Comparison of our results with the review of the literature shows a higher incidence (4- fold) of gonosomal, in particular, numerical gonosomal, chromosomal anomalies. Cytogenetic analysis is strongly suggested for infertile men, particularly in those who suffer from azoospermia.     

Inbreeding as a cause for deafness: Dadhkai study

BACKGROUND:

We report on the higher prevalence of deaf-mutes from a village in Jammu and Kashmir State of India.

MATERIALS AND METHODS:

A cross-sectional study among 79 deaf mutes using pedigree analysis, audiometry, imaging and molecular analysis.

RESULTS:

A high rate of hereditary deafness with 79 individuals diagnosed to be suffering from non-syndrome deafness in a total population of 2452 individuals residing in the village.

INTERPRETATION:

Flourishing of intermarriages led to a population with high prevalence of deafness     

Mosaic triple X syndrome in a female with primary amenorrhea

BACKGROUND:

Turner''s syndrome is the most common chromosomal abnormality in females, affecting 1 in 2,500 live female births. It is a result of absence of an X chromosome or the presence of a structurally abnormal X chromosome. Its most consistent clinical features are short stature and ovarian failure.

AIM:

The aim of the study was to report a rare case of mosaic triple X syndrome in a female with primary amenorrhea.

MATERIALS AND METHODS:

The chromosomal analysis using GTG banding was carried out, which revealed a mosaicism with 45,XO/47,XXX chromosomal constitution. Fluorescent in situ hybridization was also carried out to further confirm the observation made in the study.

CONCLUSION:

The physical features presented by the female could be due to the 45,XO/47,XXX mosaicism and the karyotype analysis was consistent with the diagnosis and clinical symptoms. Triple X mosaicism was confirmed with conventional and molecular cytogenetic analysis.     

Cost-effectiveness analysis for triple markers serum screening for Down's syndrome in Thai setting

BACKGROUND:

Down''s syndrome is an important congenital chromosomal disorder that can be seen around the world. The antenatal screening for this disorder is an important processing in present obstetrics.

OBJECTIVE:

Due to the concept of first do no harm, the use of noninvasive test is recommended. The triple marker screening test has been introduced for a few years and acceptable for its efficacy.

RESULT:

However, an important concern is on its cost-effectiveness. Here, the author analyze and present the cost-effectiveness of the triple markers serum screening for Down''s syndrome in Thai setting.

CONCLUSION:

According to this work, the cost per effectiveness of triple markers serum screening is slightly lower than standard amniocentesis test.     

A cytogenetic study of couples with recurrent spontaneous abortions and infertile patients with recurrent IVF/ICSI failure

PURPOSE:

This study was conducted to determine the frequency and contribution of chromosomal abnormalities in miscarriages and in couples with recurrent in vitro fertilization/intra cytoplasmic sperm injection (IVF/ICSI) failure.

MATERIALS and METHODS:

A total of 221 individuals; 79 with three or more recurrent spontaneous abortions and 142 with at least three IVF/ICSI failures. Chromosomal analysis from peripheral blood lymphocytes was performed according to standard cytogenetic methods using G-banding technique.

RESULTS:

Abnormal karyotype was found in 21 (9.50%) individuals. Of these 21 subjects, 4 (19.04%) exhibited sex chromosomal abnormalities and 17 (80.96%) had autosomal abnormalities. Male partners had significantly higher chromosomal abnormalities (5.88%) than of females (3.61%). These abnormalities were also higher in patients with recurrent spontaneous abortions than with IVF/ICSI failure (P < 0.05).

CONCLUSIONS:

These data may be indicative that chromosomal abnormalities are involved more in spontaneous abortions than in recurrent IVF/ICSI failure. Cytogenetic analysis could be valuable for these couples when clinical data fail to clarify the cause.     

Evaluation of concentration and storage effects of mitomycin C in the diagnosis of Fanconi anemia among idiopatic aplastic anemia patients

BACKGROUND:

Fanconi anemia (FA) is a rare autosomal recessive genetic disorder that shows an increased sensitivity to the intercalating agents such as mytomycin C (MMC), measured as chromosomal aberrations. This study was conducted to differentiate between FA and “idiopathic” aplastic anemia on the basis of induced chromosomal breakage study with MMC.

MATERIALS AND METHODS:

MMC stress tests in different final concentrations of 20 and 50 ng/ml of MMC were conducted on peripheral blood lymphocytes from 32 patients with aplastic anemia and 13 healthy controls. Fifty nanograms per milliliter of MMC from old, fresh and frozen stocks was used to check the sensitivity of diagnosis on FA-diagnosed patients. Statistical analysis was used for the assessment of aberrations, including chromatid and chromosome breaks and exchanges.

RESULTS:

Eight patients (25%) with a very high percentage of chromosomal breakage were diagnosed as FA on the basis of the chromosomal breakage study. Six of these patients exhibited congenital anomalies at presentation, while another two lacked such anomalies or had minor physical problems. Freshly made MMC has shown more sensitivity to detect FA patients compared with frozen or 1-week-old MMC stock.

CONCLUSIONS:

The study indicates that freshly made MMC stress test provides an unequivocal means of differentiation between FA and “idiopathic” aplastic anemia. Further, the study, the first of its kind from Iran, stresses on the need for conducting this test in all aplastic anemia cases, even those without congenital anomalies, for accurate and timely diagnosis of FA to implement appropriate therapy.     

Cytogenetic and comorbidity profile of Down syndrome in Mansoura University Children's Hospital, Egypt

BACKGROUND:

Down syndrome (DS) is the most common chromosomal disorder. It has three chromosomal patterns.

AIM:

To determine the cytogenetic and comorbidity profiles of DS in the Genetic Unit of Mansoura University Children''s Hospital, Mansoura, Egypt.

MATERIALS AND METHODS:

A retrospective analysis was performed on the case records of 712 cytogenetically diagnosed cases of DS at the Genetic Unit of Mansoura University Children''s Hospital, Egypt, during a 10-year period.

RESULTS:

About 19% of the cases had one or more cardiac anomalies and about 8% were hypothyroid. Nondisjunction was the most common type of abnormality, followed by translocation and lastly mosaic: 96.1, 3.1, and 0.8%, respectively. Hypothyroidism was significantly more common in translocation and mosaic karyotypes than in the nondisjunction karyotypes. First and second birth orders were significantly higher in the translocation and mosaic groups than in the nondisjunction group. Mothers are significantly older at the index pregnancy in the nondisjunction group than in the other two groups. We compared our findings with those of previous studies.

CONCLUSION:

Knowing karyotype of DS will help in genetic counseling of the parents. Wide-scale national community-based survey with DS registry could help in estimating the size of the problem.     

Prevalence of methylenetetrahydrofolate reductase 677 C-T polymorphism among mothers of Down syndrome children

INTRODUCTION:

The relationship between chromosomal non-disjunction leading to aneuploidy and folate metabolism has drawn attention in the recent years. In this study, we examined the polymorphism in the gene encoding the folate metabolizing enzyme methylenetetrahydrofolate reductase (MTHFR), namely, 677 C-T in women having Down syndrome (DS) children.

MATERIALS AND METHODS:

The prevalence of these variant genotypes (MTHFR 677 C-T polymorphism) in women having DS children (case mothers) (n = 110) was compared with controls (n = 111) from Punjab. Genotyping was done using the polymerase chain reaction method followed by restriction fragment length polymorphism.

RESULTS:

In the present study, 1.8% of case mothers had TT genotype while none of the control mothers showed this genotype. T allele frequency among cases was 0.13 and 0.11 in controls. The Chi-square value showed a non-significant difference between cases and controls.

CONCLUSION:

No association has been observed between 677 C-T polymorphism and risk of non-disjunction in case mothers. Detection of polymorphisms in more genes of folate pathway is required to find out the exact cause of non-disjunction.     

Association of microsatellite instability and chronic obstructive pulmonary disorder in isocyanate-Exposed population of Bhopal

CONTEXT:

Survivors of the Bhopal gas disaster still suffer from various respiratory ailments. We examined the effects of exposures among a cross-section of current residents suffering from COPD by ISSR-PCR.

AIMS:

Molecular screening of the gas-affected population of Bhopal with COPD for microsatellite instability due to exposure of MIC.

SETTINGS AND DESIGN:

The isocyanate-exposed population of Bhopal city suffering from chronic obstructive pulmonary disorder.

MATERIALS AND METHODS:

Inter-(SSR) analysis was used to characterize microsatellite instability in 52 MIC victims of Bhopal, suffering from COPD using (CA)₈RG and (CA)₈R[Y-Q] primer.

STATISTICAL ANALYSIS USED:

Association analyses were performed using regression analysis.

RESULTS:

The study on the MIC-affected population in Bhopal showed weak association between microsatellite instability and age (r = + 0.37); exposure distance from site (r = −0.44); and smoking status(r = + 0.12); while regression analysis of the above parameters displayed supporting evidence.

CONCLUSIONS:

The high prevalence of smoking coupled with aging and poor living habits threatens, to further increase COPD incidences among this population, highlighting the need for enhanced screening efforts.     

Chromosomal abnormalities and hormonal disorders of primary amenorrhea patients in Egypt

BACKGROUND:

Primary amenorrhea is defined as the absence of menstruation and secondary sexual characteristics in phenotypic women aged 14 years or older. Hormonal disorders are main causes of primary amenorrhea. Common hormonal cause of primary amenorrhea includes pituitary dysfunction and absent ovarian function. The aim of this study was to estimate the incidence and types of chromosomal abnormalities in patients with primary amenorrhea in Egypt.

MATERIALS AND METHODS:

Chromosomal analysis and hormonal assay were carried out on 223 patients with primary amenorrhea that were referred from different parts of Egypt to Cytogenetic laboratory of Genetic Unit, Children Hospital Mansoura University, from July 2008 to December 2010. FISH technique was carried out in some of cases to more evaluation.

RESULTS:

The frequency of chromosomal abnormalities was 46 (20.63%) in primary amenorrhea patients. The chromosomal abnormalities can be classified into four main types. (1) The numerical abnormalities of the X chromosome were detected in 23 (50 %). (2) Structural abnormalities of the X chromosome were detected in 11 (23.91%). (3) Mosaicism of X chromosome was found in 10 (21.74%). (4) Male karyotype 46, XY was presented in 2 (4.35%).

CONCLUSION:

The present study showed that karyotype and FISH are necessary to detect the causes of primary amenorrhea. This study also revealed the incidence of chromosomal abnormalities in women with primary amenorrhea in Egypt is similar to that reported in previous literatures.     

Deoxyribonucleic acid damage study in primary amenorrhea by comet assay and karyotyping

AIM:

This study aims at evaluating the chromosomal abnormalities and deoxyribonucleic acid (DNA) damage in cases with primary amenorrhea by karyotyping and comet assay.

STUDY DESIGN:

A total of 30 cases of primary amenorrhea were recruited. Secondary sexual characters were assessed by Tanner staging. Chromosomal analysis was performed by conventional phytohemagglutinin stimulated lymphocyte cell culture technique. Alkaline version of comet assay was used to evaluate DNA damage.

RESULTS:

The chromosomal pattern of 20 subjects (66.7%) was found to be normal (46,XX). Two subjects had 46,XY pattern and eight subjects had Turner syndrome (45,X or 45,X/46,XX). The comet parameters were found to be increased among subjects with 45,X monosomy, when compared to the rest of the study group and also in subjects with Tanner stage 1 when compared to stage 2.

CONCLUSION:

Comet assay revealed increased DNA damage in cases with 45,X monosomy, compared with subjects with 46,XX and 46,XY karyotype, which correlated with clinical features.     

Rapid detection of chromosome X,Y, 13, 18, and 21 aneuploidies by primed in situ labeling/synthesis technique

AIMS AND OBJECTIVE:

Primed in situ labeling/synthesis (PRINS) technique is an alternative to fluorescent in situ hybridization for chromosome analysis. This study was designed to evaluate the application of PRINS for rapid diagnosis of common chromosomal aneuploidy.

MATERIALS AND METHODS:

We have carried out PRINS using centromere specific oligonucleotide primers for chromosome X, Y, 13, 18 and 21 on lymphocyte metaphase and interphase cells spread. Specific primer was annealed in situ, followed by elongation of primer by Taq DNA polymerase in presence of labeled nucleotides. Finally, reaction was stopped and visualized directly under fluorescent microscope.

RESULTS:

Discrete centromere specific signals were observed with each primer.

CONCLUSION:

PRINS seems to be a rapid and reliable method to detect common chromosome aneuploidy in peripheral blood lymphocyte metaphase and interphase cells.