Journal of Clinical and Diagnostic Research, ISSN - 0973 - 709X

Users Online : 71815

AbstractMaterial and MethodsResultsDiscussionAcknowledgementReferences
Article in PDF How to Cite Citation Manager Readers' Comments (0) Audio Visual Article Statistics Link to PUBMED Print this Article Send to a Friend
Advertisers Access Statistics Resources

Dr Bhanu K Bhakhri

"The Journal of Clinical and Diagnostic Research (JCDR) has been in operation since almost a decade. It has contributed a huge number of peer reviewed articles, across a spectrum of medical disciplines, to the medical literature.
Its wide based indexing and open access publications attracts many authors as well as readers
For authors, the manuscripts can be uploaded online through an easily navigable portal, on other hand, reviewers appreciate the systematic handling of all manuscripts. The way JCDR has emerged as an effective medium for publishing wide array of observations in Indian context, I wish the editorial team success in their endeavour"



Dr Bhanu K Bhakhri
Faculty, Pediatric Medicine
Super Speciality Paediatric Hospital and Post Graduate Teaching Institute, Noida
On Sep 2018




Dr Mohan Z Mani

"Thank you very much for having published my article in record time.I would like to compliment you and your entire staff for your promptness, courtesy, and willingness to be customer friendly, which is quite unusual.I was given your reference by a colleague in pathology,and was able to directly phone your editorial office for clarifications.I would particularly like to thank the publication managers and the Assistant Editor who were following up my article. I would also like to thank you for adjusting the money I paid initially into payment for my modified article,and refunding the balance.
I wish all success to your journal and look forward to sending you any suitable similar article in future"



Dr Mohan Z Mani,
Professor & Head,
Department of Dematolgy,
Believers Church Medical College,
Thiruvalla, Kerala
On Sep 2018




Prof. Somashekhar Nimbalkar

"Over the last few years, we have published our research regularly in Journal of Clinical and Diagnostic Research. Having published in more than 20 high impact journals over the last five years including several high impact ones and reviewing articles for even more journals across my fields of interest, we value our published work in JCDR for their high standards in publishing scientific articles. The ease of submission, the rapid reviews in under a month, the high quality of their reviewers and keen attention to the final process of proofs and publication, ensure that there are no mistakes in the final article. We have been asked clarifications on several occasions and have been happy to provide them and it exemplifies the commitment to quality of the team at JCDR."



Prof. Somashekhar Nimbalkar
Head, Department of Pediatrics, Pramukhswami Medical College, Karamsad
Chairman, Research Group, Charutar Arogya Mandal, Karamsad
National Joint Coordinator - Advanced IAP NNF NRP Program
Ex-Member, Governing Body, National Neonatology Forum, New Delhi
Ex-President - National Neonatology Forum Gujarat State Chapter
Department of Pediatrics, Pramukhswami Medical College, Karamsad, Anand, Gujarat.
On Sep 2018




Dr. Kalyani R

"Journal of Clinical and Diagnostic Research is at present a well-known Indian originated scientific journal which started with a humble beginning. I have been associated with this journal since many years. I appreciate the Editor, Dr. Hemant Jain, for his constant effort in bringing up this journal to the present status right from the scratch. The journal is multidisciplinary. It encourages in publishing the scientific articles from postgraduates and also the beginners who start their career. At the same time the journal also caters for the high quality articles from specialty and super-specialty researchers. Hence it provides a platform for the scientist and researchers to publish. The other aspect of it is, the readers get the information regarding the most recent developments in science which can be used for teaching, research, treating patients and to some extent take preventive measures against certain diseases. The journal is contributing immensely to the society at national and international level."



Dr Kalyani R
Professor and Head
Department of Pathology
Sri Devaraj Urs Medical College
Sri Devaraj Urs Academy of Higher Education and Research , Kolar, Karnataka
On Sep 2018




Dr. Saumya Navit

"As a peer-reviewed journal, the Journal of Clinical and Diagnostic Research provides an opportunity to researchers, scientists and budding professionals to explore the developments in the field of medicine and dentistry and their varied specialities, thus extending our view on biological diversities of living species in relation to medicine.
‘Knowledge is treasure of a wise man.’ The free access of this journal provides an immense scope of learning for the both the old and the young in field of medicine and dentistry as well. The multidisciplinary nature of the journal makes it a better platform to absorb all that is being researched and developed. The publication process is systematic and professional. Online submission, publication and peer reviewing makes it a user-friendly journal.
As an experienced dentist and an academician, I proudly recommend this journal to the dental fraternity as a good quality open access platform for rapid communication of their cutting-edge research progress and discovery.
I wish JCDR a great success and I hope that journal will soar higher with the passing time."



Dr Saumya Navit
Professor and Head
Department of Pediatric Dentistry
Saraswati Dental College
Lucknow
On Sep 2018




Dr. Arunava Biswas

"My sincere attachment with JCDR as an author as well as reviewer is a learning experience . Their systematic approach in publication of article in various categories is really praiseworthy.
Their prompt and timely response to review's query and the manner in which they have set the reviewing process helps in extracting the best possible scientific writings for publication.
It's a honour and pride to be a part of the JCDR team. My very best wishes to JCDR and hope it will sparkle up above the sky as a high indexed journal in near future."



Dr. Arunava Biswas
MD, DM (Clinical Pharmacology)
Assistant Professor
Department of Pharmacology
Calcutta National Medical College & Hospital , Kolkata




Dr. C.S. Ramesh Babu
" Journal of Clinical and Diagnostic Research (JCDR) is a multi-specialty medical and dental journal publishing high quality research articles in almost all branches of medicine. The quality of printing of figures and tables is excellent and comparable to any International journal. An added advantage is nominal publication charges and monthly issue of the journal and more chances of an article being accepted for publication. Moreover being a multi-specialty journal an article concerning a particular specialty has a wider reach of readers of other related specialties also. As an author and reviewer for several years I find this Journal most suitable and highly recommend this Journal."
Best regards,
C.S. Ramesh Babu,
Associate Professor of Anatomy,
Muzaffarnagar Medical College,
Muzaffarnagar.
On Aug 2018




Dr. Arundhathi. S
"Journal of Clinical and Diagnostic Research (JCDR) is a reputed peer reviewed journal and is constantly involved in publishing high quality research articles related to medicine. Its been a great pleasure to be associated with this esteemed journal as a reviewer and as an author for a couple of years. The editorial board consists of many dedicated and reputed experts as its members and they are doing an appreciable work in guiding budding researchers. JCDR is doing a commendable job in scientific research by promoting excellent quality research & review articles and case reports & series. The reviewers provide appropriate suggestions that improve the quality of articles. I strongly recommend my fraternity to encourage JCDR by contributing their valuable research work in this widely accepted, user friendly journal. I hope my collaboration with JCDR will continue for a long time".



Dr. Arundhathi. S
MBBS, MD (Pathology),
Sanjay Gandhi institute of trauma and orthopedics,
Bengaluru.
On Aug 2018




Dr. Mamta Gupta,
"It gives me great pleasure to be associated with JCDR, since last 2-3 years. Since then I have authored, co-authored and reviewed about 25 articles in JCDR. I thank JCDR for giving me an opportunity to improve my own skills as an author and a reviewer.
It 's a multispecialty journal, publishing high quality articles. It gives a platform to the authors to publish their research work which can be available for everyone across the globe to read. The best thing about JCDR is that the full articles of all medical specialties are available as pdf/html for reading free of cost or without institutional subscription, which is not there for other journals. For those who have problem in writing manuscript or do statistical work, JCDR comes for their rescue.
The journal has a monthly publication and the articles are published quite fast. In time compared to other journals. The on-line first publication is also a great advantage and facility to review one's own articles before going to print. The response to any query and permission if required, is quite fast; this is quite commendable. I have a very good experience about seeking quick permission for quoting a photograph (Fig.) from a JCDR article for my chapter authored in an E book. I never thought it would be so easy. No hassles.
Reviewing articles is no less a pain staking process and requires in depth perception, knowledge about the topic for review. It requires time and concentration, yet I enjoy doing it. The JCDR website especially for the reviewers is quite user friendly. My suggestions for improving the journal is, more strict review process, so that only high quality articles are published. I find a a good number of articles in Obst. Gynae, hence, a new journal for this specialty titled JCDR-OG can be started. May be a bimonthly or quarterly publication to begin with. Only selected articles should find a place in it.
An yearly reward for the best article authored can also incentivize the authors. Though the process of finding the best article will be not be very easy. I do not know how reviewing process can be improved. If an article is being reviewed by two reviewers, then opinion of one can be communicated to the other or the final opinion of the editor can be communicated to the reviewer if requested for. This will help one’s reviewing skills.
My best wishes to Dr. Hemant Jain and all the editorial staff of JCDR for their untiring efforts to bring out this journal. I strongly recommend medical fraternity to publish their valuable research work in this esteemed journal, JCDR".



Dr. Mamta Gupta
Consultant
(Ex HOD Obs &Gynae, Hindu Rao Hospital and associated NDMC Medical College, Delhi)
Aug 2018




Dr. Rajendra Kumar Ghritlaharey

"I wish to thank Dr. Hemant Jain, Editor-in-Chief Journal of Clinical and Diagnostic Research (JCDR), for asking me to write up few words.
Writing is the representation of language in a textual medium i e; into the words and sentences on paper. Quality medical manuscript writing in particular, demands not only a high-quality research, but also requires accurate and concise communication of findings and conclusions, with adherence to particular journal guidelines. In medical field whether working in teaching, private, or in corporate institution, everyone wants to excel in his / her own field and get recognised by making manuscripts publication.


Authors are the souls of any journal, and deserve much respect. To publish a journal manuscripts are needed from authors. Authors have a great responsibility for producing facts of their work in terms of number and results truthfully and an individual honesty is expected from authors in this regards. Both ways its true "No authors-No manuscripts-No journals" and "No journals–No manuscripts–No authors". Reviewing a manuscript is also a very responsible and important task of any peer-reviewed journal and to be taken seriously. It needs knowledge on the subject, sincerity, honesty and determination. Although the process of reviewing a manuscript is a time consuming task butit is expected to give one's best remarks within the time frame of the journal.
Salient features of the JCDR: It is a biomedical, multidisciplinary (including all medical and dental specialities), e-journal, with wide scope and extensive author support. At the same time, a free text of manuscript is available in HTML and PDF format. There is fast growing authorship and readership with JCDR as this can be judged by the number of articles published in it i e; in Feb 2007 of its first issue, it contained 5 articles only, and now in its recent volume published in April 2011, it contained 67 manuscripts. This e-journal is fulfilling the commitments and objectives sincerely, (as stated by Editor-in-chief in his preface to first edition) i e; to encourage physicians through the internet, especially from the developing countries who witness a spectrum of disease and acquire a wealth of knowledge to publish their experiences to benefit the medical community in patients care. I also feel that many of us have work of substance, newer ideas, adequate clinical materials but poor in medical writing and hesitation to submit the work and need help. JCDR provides authors help in this regards.
Timely publication of journal: Publication of manuscripts and bringing out the issue in time is one of the positive aspects of JCDR and is possible with strong support team in terms of peer reviewers, proof reading, language check, computer operators, etc. This is one of the great reasons for authors to submit their work with JCDR. Another best part of JCDR is "Online first Publications" facilities available for the authors. This facility not only provides the prompt publications of the manuscripts but at the same time also early availability of the manuscripts for the readers.
Indexation and online availability: Indexation transforms the journal in some sense from its local ownership to the worldwide professional community and to the public.JCDR is indexed with Embase & EMbiology, Google Scholar, Index Copernicus, Chemical Abstracts Service, Journal seek Database, Indian Science Abstracts, to name few of them. Manuscriptspublished in JCDR are available on major search engines ie; google, yahoo, msn.
In the era of fast growing newer technologies, and in computer and internet friendly environment the manuscripts preparation, submission, review, revision, etc and all can be done and checked with a click from all corer of the world, at any time. Of course there is always a scope for improvement in every field and none is perfect. To progress, one needs to identify the areas of one's weakness and to strengthen them.
It is well said that "happy beginning is half done" and it fits perfectly with JCDR. It has grown considerably and I feel it has already grown up from its infancy to adolescence, achieving the status of standard online e-journal form Indian continent since its inception in Feb 2007. This had been made possible due to the efforts and the hard work put in it. The way the JCDR is improving with every new volume, with good quality original manuscripts, makes it a quality journal for readers. I must thank and congratulate Dr Hemant Jain, Editor-in-Chief JCDR and his team for their sincere efforts, dedication, and determination for making JCDR a fast growing journal.
Every one of us: authors, reviewers, editors, and publisher are responsible for enhancing the stature of the journal. I wish for a great success for JCDR."



Thanking you
With sincere regards
Dr. Rajendra Kumar Ghritlaharey, M.S., M. Ch., FAIS
Associate Professor,
Department of Paediatric Surgery, Gandhi Medical College & Associated
Kamla Nehru & Hamidia Hospitals Bhopal, Madhya Pradesh 462 001 (India)
E-mail: drrajendrak1@rediffmail.com
On May 11,2011




Dr. Shankar P.R.

"On looking back through my Gmail archives after being requested by the journal to write a short editorial about my experiences of publishing with the Journal of Clinical and Diagnostic Research (JCDR), I came across an e-mail from Dr. Hemant Jain, Editor, in March 2007, which introduced the new electronic journal. The main features of the journal which were outlined in the e-mail were extensive author support, cash rewards, the peer review process, and other salient features of the journal.
Over a span of over four years, we (I and my colleagues) have published around 25 articles in the journal. In this editorial, I plan to briefly discuss my experiences of publishing with JCDR and the strengths of the journal and to finally address the areas for improvement.
My experiences of publishing with JCDR: Overall, my experiences of publishing withJCDR have been positive. The best point about the journal is that it responds to queries from the author. This may seem to be simple and not too much to ask for, but unfortunately, many journals in the subcontinent and from many developing countries do not respond or they respond with a long delay to the queries from the authors 1. The reasons could be many, including lack of optimal secretarial and other support. Another problem with many journals is the slowness of the review process. Editorial processing and peer review can take anywhere between a year to two years with some journals. Also, some journals do not keep the contributors informed about the progress of the review process. Due to the long review process, the articles can lose their relevance and topicality. A major benefit with JCDR is the timeliness and promptness of its response. In Dr Jain's e-mail which was sent to me in 2007, before the introduction of the Pre-publishing system, he had stated that he had received my submission and that he would get back to me within seven days and he did!
Most of the manuscripts are published within 3 to 4 months of their submission if they are found to be suitable after the review process. JCDR is published bimonthly and the accepted articles were usually published in the next issue. Recently, due to the increased volume of the submissions, the review process has become slower and it ?? Section can take from 4 to 6 months for the articles to be reviewed. The journal has an extensive author support system and it has recently introduced a paid expedited review process. The journal also mentions the average time for processing the manuscript under different submission systems - regular submission and expedited review.
Strengths of the journal: The journal has an online first facility in which the accepted manuscripts may be published on the website before being included in a regular issue of the journal. This cuts down the time between their acceptance and the publication. The journal is indexed in many databases, though not in PubMed. The editorial board should now take steps to index the journal in PubMed. The journal has a system of notifying readers through e-mail when a new issue is released. Also, the articles are available in both the HTML and the PDF formats. I especially like the new and colorful page format of the journal. Also, the access statistics of the articles are available. The prepublication and the manuscript tracking system are also helpful for the authors.
Areas for improvement: In certain cases, I felt that the peer review process of the manuscripts was not up to international standards and that it should be strengthened. Also, the number of manuscripts in an issue is high and it may be difficult for readers to go through all of them. The journal can consider tightening of the peer review process and increasing the quality standards for the acceptance of the manuscripts. I faced occasional problems with the online manuscript submission (Pre-publishing) system, which have to be addressed.
Overall, the publishing process with JCDR has been smooth, quick and relatively hassle free and I can recommend other authors to consider the journal as an outlet for their work."



Dr. P. Ravi Shankar
KIST Medical College, P.O. Box 14142, Kathmandu, Nepal.
E-mail: ravi.dr.shankar@gmail.com
On April 2011
Anuradha

Dear team JCDR, I would like to thank you for the very professional and polite service provided by everyone at JCDR. While i have been in the field of writing and editing for sometime, this has been my first attempt in publishing a scientific paper.Thank you for hand-holding me through the process.


Dr. Anuradha
E-mail: anuradha2nittur@gmail.com
On Jan 2020

Important Notice

Original article / research
Year : 2009 | Month : February | Volume : 3 | Issue : 1 | Page : 1266 - 1273

Screening For Fragile X Syndrome Among Neurobehavioural Patients From Kolkata, Eastern India.

DAS B *, DUTTA S** , CHATTERJEE A***, SINHA S****,CHATTOPADHYAY A*****, MUKHOPADHYAY K******

*(M.SC),**(M.SC),****(MBBS),***(M.Sc),*****(Ph.D),******(Ph.D)Manovikas Biomedical Research andDiagnostic Centre,Kolkata-700107,(India).

Correspondence Address :
Dr.Kanchan Mukhopadhyay,Manovikas Biomedical Research and Diagnostic Centre 482,Madudah,Plot I-24,Sec J,E.M.Bypass,Kolkata-700107,(India)Ph:91-33-4001-9179;Fax: 91-33-2442-8275,Email:kanchanmvk@yahoo.com

Abstract

Background: Fragile X syndrome (FXS), associated with abnormal functioning of the FMR1 gene, is a major cause for inherited mental retardation (MR). The symptoms which are commonly associated with FXS are also observed in patients suffering from various neuropsychiatric disorders like autism, epilepsy, seizure disorder etc. Thus, the diagnosis of FXS that is solely based on a patient’s physical and behavioural characteristics is very difficult. To avoid a false positive diagnosis which is crucial for better management of the disorder, screening for FXS with easy diagnostic tools becomes extremely important.
Aims: In this study, screening for FXS was carried out among 157 various neurobehavioural patients attending the out patients department of Manovikas Kendra, Kolkata.
Methods and Material: To screen the level of functioning of the FMR1 gene, the percentage of leukocytes expressing the fragile X mental retardation protein (FMRP) was measured by an immunocytochemical method. CGG repeat size was analyzed by PCR amplification and FMR1 promoter methylation status was checked by methylation sensitive-PCR.
Results: Out of 157 patients recruited for this study, only four were confirmed as FXS (3.18% prevalence among neurobehavioural outpatients). 30 distinct alleles with 12-49 CGG repeats were detected, with the 27 and 28 repeats being most common. Premutation alleles were observed in 25 subjects. Molecular biology-based analyses confirmed 5 cases as FXS; four patients were detected with promoter methylation mosaicism and one with full methylation.
Conclusion: In the present investigation, FXS screening was performed on various neurobehavioural outpatients and four were confirmed with the disorder. The CGG repeat alleles that were most frequently observed in this study were different from those found in other studies, indicating a racial or ethnic variation.

Keywords

Fragile X syndrome, MR, FMRP, CGG repeat, MS-PCR.

Introduction
Fragile X syndrome (MIM 309550) is one of the monogenic forms of X-linked mental retardation (MR), with a worldwide prevalence of 1 in 4000 males and 1 in 8000 females among the western population (1),(2),(3). This is the common genetic cause for MR, second only to Down syndrome (DS). Studies carried out on Indian male MR cases revealed ~7% possibility of having FXS (4),(5),(6),(7).

The primary features of FXS are mild to severe MR, long narrow face, large ears, broad nasal bridge, prominent jaw, hyperextensible finger joints, double jointed thumbs, single palmer crease, flat feet, mitral valve prolapse, velvet-like skin and macroorchidism (8),(9),(10). General growth abnormalities i.e. increased height throughout childhood and early onset of puberty as compared to peers is also observed in affected male individuals. Other medical problems like recurrent emesis, otitis media infections, sinusitis, strabismus, and seizures are also associated with FXS (11), (12). Patients often display autistic features such as difficulties in social interaction with peers, impaired verbal and nonverbal communication, poor eye contact, tactile defensiveness, hand flapping, hand biting and other stereotypic movements and it is estimated that nearly 25% of children with FXS meet these criteria for autism (13). A majority of FXS children also have problems with attention and hyperactivity; deficit in attention alongwith hyperactivity in boys with FXS has been reported to vary between 70 to 100% in various studies (14),(15),(16). Some other associated features are hyperarousal, social avoidance, anxiety, tantrums, extreme sensitivity to sensations and sometimes, aggression. Epilepsy is also reported to occur in 10 to 20% of individuals having FXS (17).

FXS and DS the two most prevalent forms of MR that are linked to faulty brain synaptic communications ( http://med.stanford.edu/news_releases/2007/april/fragilex.html ). A similar kind of study, earlier done by Dr. Daniel Madison on murine model of FXS and DS, showed that not only the post-synaptic cells, but also the pre-synaptic cells are important ones to study in these disorders (18),(19).

FXS is mostly caused by a mutation in the FMR1 gene which is located on the X chromosome, approximately 38 kb in size and contains 17 exons (20), (21).The FMR1 gene product, fragile X mental retardation protein (FMRP), is an RNA-binding protein that is expressed in foetal and adult tissue cytoplasm, with the most abundant expression in brain and testes (22). It plays an important role in the regulation of specific target mRNA translation in the cell cytoplasm (23). FMRP is also involved in the regulation of the synaptic activity and loss of this protein directly influences neuronal plasticity, resulting in cognitive defects (24). An expansion of a trinucleotide (CGG) repeat at the highly conserved 5' UTR of the FMR1 gene causes instability; size of the CGG repeats often increases during female meiosis in succeeding generations (25). Generally, normal subjects have 2-50 copies of the repeat and those having the repeat number in between 50-200 are known as carriers, also known as premutation (PreM) condition (26). Hypermethylation of both the repeat and its adjacent promoter region, concomitant with expansion of the CGG repeats (>200-220 repeats), leads to a decrease in the transcription of FMR1; a condition known as full mutation (FM). The number of repeats in the PreM condition is potentially unstable and may expand into the FM range. Although a vast majority of patients with FXS show this pattern of expanded repeats, a small number of cases have also been reported where partial deletions

For detection of FXS, methods like Southern-blot analysis, polymerase chain reaction (PCR) and immunocytochemical analyses are used (28),(29),(30),(31). The most commonly applied molecular genetic technique depends on either the detection of expanded repeats or de novo methylation analysis, or both. Although Southern hybridization is a very popular and probably the most reliable method for detection of FXS, it has several disadvantages for rapid screening. The main disadvantage is that it is a time consuming and laborious process and hence, is disadvantageous for the rapid screening of a large population. Moreover, it is expensive and requires large amount of DNA. Because of these limitations, for rapid screening, other diagnostic tests like repeat PCR analysis and methylation-sensitive PCR (MS-PCR) analysis for genomic DNA (29),(30) and an immunocytochemical technique for detection of FMRP (31),(32) were developed.

For the present investigation, population screening for FXS was performed among individuals living in and around Kolkata, West Bengal, who attended the psychiatric out-patient department for various neurobehavioural disorders like autism , spectrum disorder (ASD), epilepsy, attention deficit hyperactivity disorder (ADHD), DS and MR. We have relied mainly on the immunocytochemical detection of FMRP, repeat PCR and MS-PCR analyses for screening.

Material and Methods

Subjects
Probands (n=157) with various neurobehavioural disorders were recruited from the out-patient department of Manovikas Kendra Rehabilitation and Research Institute for the Handicapped, Kolkata, India. Diagnosis was carried out by mental health professionals according to the DSM-IV criteria (33). Among the 157 patients, 71.34% were male and 28.66% were female, with age ranges of 1.5-24 yrs and 1-35 yrs, respectively. Study subjects comprised of ADHD (n=16), ASD (n=33), Rett syndrome (n=1), epilepsy (n=2), DS (n=35) and MR (n=70); among the MR cases, 22 exhibited symptoms of FXS.

Sample Collection
Peripheral blood was collected in an anticoagulant from probands and their parents after obtaining their informed written consent. Samples collected in heparin were used for the detection of FMRP and chromosomal abnormalities. Leukocytes cultured in regular as well as folate-deficient RPMI 1640 media (34) were processed for GTG-banding and karyotype analysis (35). The protocol was approved by the Institutional Human Ethical committee.

Genomic DNA extracted from leucocytes using the standard protocol (36), was used for PCR-based diagnosis of genetic abnormalities (amplification of CGG repeat and MS-PCR analysis).

Detection of FMRP
Intracellular FMRP was detected by an immunocytochemical technique (31). In summary, peripheral blood smears on glass slides were collected and stored at -20°C till the assay was done (a maximum of 2 wks). Before staining, the smear was fixed with 3% paraformaldehyde (Sigma, USA) in phosphate buffer (pH 7.3) and permeabilized with methanol (SRL, India). Incubations with the 1st antibody (goat FMRP-specific polyclonal IgG, Santa-Cruz Biotechnology, USA) and 2nd antibody (rabbit anti-goat antibody coupled with FITC, Santa-Cruz Biotechnology, USA) were carried out for 2hrs and 1hr, respectively, at room temperature. Cells were viewed under Zeiss Axioskop 2 plus fluorescence microscope. For each sample, nearly 1000 cells were scored and the percentage of lymphocytes expressing FMRP was determined.

Determination Of CGG Repeat Number
Primers used for amplification of the CGG repeat region of FMR1 gene were designed in the lab, using the Primer3 software; primer sequences and PCR conditions will be available on request. PCR amplicons were separated by 12% polyacrylamide gel electrophoresis, visualized by UV fluorescence following ethidium bromide staining and analyzed using Quantity One software. Using this system, repeats in the lower range (<180) could be easily detected; however, those in the higher range (~200-250 repeats) were not detectable.

Determination Of FMR1 Gene Promoter Methylation Status
Genomic DNA (~5 μg) was deaminated using the EZ DNA methylation kit (Zymo research, USA). Methylation-sensitive PCR (MS-PCR) was carried out by following the method of Weinhäusel and Haas (29). Multiplex PCR for simultaneous amplification of the FMR1 and Xist (X-Inactive Specific Transcript) promoter sequences was performed. Amplification was carried out in a final reaction volume of 20° mL containing 75 ng of sodium bisulfite-treated DNA, 1.0 U Taq polymerase (Bangalore Genei, India), 0.2 mM dNTP mix (Bangalore Genei, India), 1X Taq buffer B (Bangalore Genei, India) and 1.5 mM MgCl2. Primer concentrations were as described by Weinhäusel et al and Haas (29). After an initial denaturation at 95°C for 5mins, amplification was performed for: 35 cycle of denaturation at 95°C for 30 sec, annealing at 60°C for 20 sec and extension at 72°C for 40 sec; cyclic reaction was followed by a final extension at 72°C for 7 mins. PCR amplicons were analyzed in a 2.5% agarose gel, followed by ethidium bromide staining and visualization under UV fluorescence.


Results

Out of the 157 cases screened, only five cases (3.18%) were confirmed as FXS, among which four were mosaic and one was homozygous for methylation of the FMR1 promoter. Details of the results are given in (Table/Fig 1).

Chromosomal Investigation
All DS cases recruited for this study showed trisomy 21. Among the MR cases, one was confirmed as Prader-Willi syndrome (deletion in 15q11-q13 region). In the FXS cases, deletion of Xq27.3 was detected in 50~54% of dividing cells (Table/Fig 2). No other chromosomal abnormalities were observed.

Result Of Immunocytochemical FMRP Expression Test
The percentage of FMRP positive cells (Table/Fig 3) varied between 10%-93% in different neurobehavioural patients. In ADHD cases, the average percentage of FMRP positive cells was found to be 37.26±19.64 (Table/Fig 1]. Among the ASD patients, the mean FMRP positive cell percentage was found to be 49.99±22.6. In DS subjects, the FMRP positive cell percentage was 44.56±19.64. Among the MR and epilepsy cases, the mean positive cell count was 43.27 and 48.14 respectively, of which 10 MR cases were found to have less than 20% positive cells. Among the MR cases, 2 were confirmed as FXS, one with full mutation and the other one with mosaicism (Table/Fig 4). Other MR subjects with 35.29% and 76.47% FMRP positive cells were confirmed as FXS mosaic. One patient with epileptic seizures was also confirmed as mosaic FXS, with 42.8% FMRP positive cells.

Repeat PCR Analysis
Primers used for the repeat PCR assay identified 136 bp of unique sequence along with the CGG repeat sequence (Table/Fig 5).

The repeat numbers varied within 12-49; the most common repeat being 27 (22.53%), followed by 28 (19.23%), 26 (7.14%) and 29 (6.59%) repeats (Table/Fig 6). Samples from men normally showed only one allele, excepting a few cases where PreM alleles were found along with the normal allele. Among the 45 women subjects, 27 females showed homozygosity for a particular type of repeat and in the remaining 18, at least two different alleles were observed. Among ADHD patients, one patient had a PreM allele (58) along with the other normal allele. In ASD patients, 9 cases were found to have PreM alleles, with repeat sizes ranging from 53-156. In case of DS, 6 cases had PreM alleles ranging from 53-172 repeats. Out of the 70 MR cases, 9 cases had PreM alleles containing 51-180 repeats. No PreM allele was detected in the two epilepsy cases (Table/Fig 1).

MS-PCR Analysis
Only those cases which had a low FMRP level (< 20%) or had repeat alleles in the PreM range, or failed to amplify during repeat analysis, were considered for promoter methylation analyses by MS-PCR. Out of the total 80 cases evaluated, five cases were confirmed as FXS, all of which were male; four of them were mosaic, having both methylated (PM) and unmethylated (PU) promoters in the FMR1, as well as in Xist. In the fifth sample, only one band for the PM was obtained (Table/Fig 7),(Table/Fig 4).


Discussion

In 1943, Martin and Bell first reported a large family with 11 mentally retarded males and a few mildly affected females that showed MR segregating as an X-linked trait (37).

The cytogenetic marker, delXq27.3, is observed in ~60% cells (38) (which were cultured in folic acid or thymidine deficient culture media)(39) of affected male individuals and as well as female carriers. Until 1991, cytogenetic testing of the fragile X site was the only means for diagnosis of FXS. In the present investigation, by karyoptype analysis, Xq27.3 deletion was observed in only 50-54% of cells of FXS subjects, confirming the previous reports.

Because of the complexity and uncertainty of the cell culture method, further development in the detection of FXS was necessitated, which led to the development of methods like Southern blotting and MS-PCR analyses (28),(29),(30). For population screening, the immunocytochemical detection of FMRP was devised (31). The drawback of the immunocytochemical method lies in the fact that, even in individuals with a full mutation, a base line FMRP is detectable. This is also evident from the present investigation, since in mosaic FXS cases, the FMRP positive cell percentage was ~40%. Therefore, the immunocytochemical method is not reliable for the diagnosis of FXS.

We have observed that both the repeat PCR and MS-PCR analysis are useful for the rapid screening of FXS and 157 neurobehavioural patients were screened using a combination of these two methods. We could detect only five men affected with FXS, one with full promoter methylation and four with methylation mosaicism, representing a frequency of 3.18%, which is consistent with the frequencies observed by Maino et al. (40) and Brown (41), which ranged from 2-7%. This result is also consistent with the frequency observed by Elango et al. (42) in Indian male patients with MR (2.8% prevalence of FXS). On the contrary, previous studies carried out on mentally retarded Indian males revealed ~7% possibility of having FXS (4),(5),(6),(7); this could be due to population differences, since the Indian population is extremely heterogeneous.

One significant observation made in this study, was the low frequency of premutation alleles (6.25%) in ADHD patients who have normal intelligence (Table/Fig 1). In the MR cases also, PreM allele frequency was comparatively lower (12.9%). No PreM allele was detected in the two epilepsy cases. On the contrary, in ASD and DS patients with neurodevelopmental deficits, PreM alleles, with repeat sizes ranging 53-172, were present in higher frequency (26.5% in ASD and 17.1% in DS). From the above observations, we conclude that premutation alleles are associated with at least moderate impairments in intellectual functioning, while it is absent in individuals with normal intelligence or mild impairment only.

Among the 45 women samples studied, seven PreM alleles were detected. Rousseau et al. (43) reported the frequency of PreM to be 1:500 X chromosomes in a large group of women samples. However, it is difficult to make such a conclusion from the present study, due to small sample size.

Among the 30 different normal CGG repeat alleles found in the present study, (CGG)27 followed by (CGG)28, appeared to be the most frequent alleles in this population. (CGG)12 was the smallest and (CGG)49 was the largest allele observed (Table/Fig 7). A report from the southern part of the India also documented (CGG)28 and (CGG)31 as the most frequent alleles (5). A study conducted on non-retarded Japanese subjects also showed (CGG)28 to be the most frequent allele (44). On the other hand, studies on Caucasians, Hispanics, Blacks, and Chinese reported (CGG)29 to be the most prevalent allele (45),(46).

Methylation of the CpG island near the CGG repeat sequence, which influences expression of the FMR1 gene, was studied by MS-PCR. (29). We have observed full methylation in only one male MR patient and methylation mosaicism in three male MR patients and one male epilepsy patient. None of the female probands were found to be affected by FXS. However, the major drawback or limitation of the MS-PCR method was its failure to detect mosaic females (29).

Based on the data presented above, we may state that the frequency of FXS among neurobehavioural outpatients is rather low (3.18%) in the studied population. However, the present study emphasizes the importance of screening neurobehavioural patients for FXS, since psychiatric evaluation alone may not be sufficient for diagnosing FXS patients. It is also stated that for screening a large number of individuals, a combination of tests would be beneficial. PCR-based techniques could be used for short listing probands, followed by Southern blot analysis for confirmation, thus saving time while making it cost effective.

Acknowledgement

Authors are thankful to all the families for their participation in the study. This work was sponsored by the Indian Council of Medical Research, Government of India, New Delhi; Grant No. 5/4-4/11/M/2003-NCD-I.

References

1.
. Crawford DC, Acuna JM, Sherman SL. FMR1 and the fragile X syndrome: Human genome epidemiology review. Genet Med 2001; 3:359–71.
2.
. Turner G, Webb T, Wake S, Robinson H. Prevalence of fragile X syndrome. Am J Med Genet 1996; 64: 196-97.
3.
. Warren ST, Nelson DI. Advances in molecular analysis of fragile X syndrome. J Am Med Assoc 1994; 271:536-42.
4.
. Jain U, Verma IC, Kapoor AK. Prevalence of fragile X (A) syndrome in mentally retarded children at a genetics referral centre in Delhi, India. Indian J Med Res, 1998; 108:12-16.
5.
. Baskaran S, Naseerullah MK, Manjunatha KR, et al. Triplet repeat polymorphism & fragile X syndrome in the Indian context. Indian J Med Res. 1998; 107:29-36.
6.
. Sharma D, Gupta M, Thelma BK. Expansion mutation frequency and CGG/GCC repeat polymorphism in FMR1 and FMR2 genes in an Indian population. Genet Epidemiol. 2001; 20:129-44.
7.
. Saha S, Karmakar P, Chatterjee C, et al. Fragile X syndrome in Calcutta, India. Ann Clin Biochem. 2001; 38:264-71.
8.
. Bennetto L, Pennington BF. The neuropsychology of fragile X syndrome. In: Hagerman RJ, Cronister A, editors. Fragile X syndrome: diagnosis, treatment and research. Baltimore: The Johns Hopkins University Press; 1996. p. 210–48.
9.
. Turner G, Daniel A, Frost M. X-linked mental retardation, macro-orchidism, and the Xq27 fragile site. J Pediatr. 1980; 96:837-41.
10.
. Hagerman RJ, Synhorst DP. Mitral valve prolapse and aortic dilatation in the fragile X syndrome. Am J Med Genet. 1984; 17:123-31.
11.
. Hagerman RJ, Altshul-Stark D, McBogg P. Recurrent otitis media in the Fragile X syndrome. Am J Dis Child. 1987; 141:184-87.
12.
. Sabaratnam M, Vroegop PG, Gangadharan SK. Epilepsy and EEG findings in 18 males with fragile X syndrome. Seizure,. 2001; 10:60-63.
13.
. Bailey DB, Mesibov G, Hatton DD, et al. Autistic behavior in young boys with fragile X syndrome. J Autism Dev Disord. 1998; 28:499–508.
14.
. Baumgardner TL, Reiss AL, Freund LS, Abrams MT. Specification of the neurobehavioral phenotype in males with fragile X syndrome. Pediatrics. 1995; 95:744-52.
15.
. Borghgraef M, Fryns JP, Dielkens A, et al. Fragile (X) syndrome: a study of the psychological profile in 23 prepubertal patients. Clin Genet . 1987; 32:179-86.
16.
. Bregman JD, Leckman JF, Ort SI. Fragile X syndrome: genetic predisposition to psychopathology. J Autism Dev Disord. 1988; 18: 343-54.
17.
. Berry-Kravis E. Epilepsy in fragile x syndrome. Dev Med and Child Neurol. 2002; 44:724-28.
18.
. Hanson JE, Blank M, Valenzuela RA, et al. The functional nature of synaptic circuitry is altered in area CA3 of the hippocampus in a mouse model of Down’s syndrome. J Physiol. 2007a; 579: 53-67.
19.
. Hanson JE, Madison DV. Presynaptic Fmr1 Genotype Influences the Degree of Synaptic Connectivity in a Mosaic Mouse Model of Fragile X Syndrome. J Neurosci 2007b; 27:4014-18.
20.
. Ashley CT Jr, Wilkinson KD, Reines D, Warren ST. FMR1 protein: conserved RNP family domains and selective RNA binding. Science 1993; 262:563-66.
21.
. Eichler EE, Richards S, Gibbs RA, Nelson DL. Fine structure of the human FMR1 gene. Hum Mol Genet, 1993; 2:1147-1153. Erratum in: Hum Mol Genet 1994; 3:684-85.
22.
. Devys D, Lutz Y, Rouyer N, et al. The FMR-1 protein is cytoplasmic, most abundant in neurons and appears normal in carriers of a fragile X premutation. Nat Genet 1993; 4:335-40.
23.
. Feng Y, Absher D, Eberhart DE, et al. FMRP associates with polyribosomes as an mRNP, and the I304N mutation of severe fragile X syndrome abolishes this association. Mol.Cell. 1997; 1:109-18.
24.
. Jin P, Warren TS. New insights into fragile X syndrome: from molecules to neurobehaviors, Trends in Biochemical Sciences 2003; 28: 152-58.
25.
. Oberle I, Rousseau F, Heitz D, et al. Instability of a 550 bas-pair DAN segment and abnormal methylation in fragile x syndrome. Science 1991; 252:1097-1102.
26.
. Verkerk AJ, Pieretti M, Sutcliffe JS, et al. Identification of a gene (FMR-1) containing CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile x syndrome. Cell 1991; 65:905-14.
27.
. Hammond LS, Macias MM, Tarleton JC, et al. Fragile X syndrome and deletions in FMR1: new case and review of the literature. Am J Med Genet 1997; 72:430-34.
28.
. Nolin SL, Brown WT, Glicksman A et al. Expansion of the Fragile X CGG repeat in females with premutation and intermediate alleles. Am. J. Hum. Genet 2003; 72:454-64.
29.
. Weinhausel A, Haas OA. Evaluation of the fragile X (FRAXA) syndrome with methylation-sensitive PCR. Hum Genet 2001; 108:450-58.
30.
. Brown WT, Houck GE Jr, Jeziorowska A, et al. Rapid fragile X carrier screening and prenatal diagnosis using a non- radioactive PCR test. J Am Med Assoc 1993; 270: 1569-75.
31.
. Willemsen R, Mohkamsing S, de Vries B, et al. Rapid antibody test for fragile X syndrome. Lancet 1995; 345:1147-48.
32.
. Willemsen R, Smits A, Mohkamsing S, et al. Rapid antibody test for diagnosing fragile X syndrome: a validation of the technique. Hum Genet 1997; 99:308-11.
33.
. American Psychiatric Association. Diagnostic and Statistical Manual for Mental Disorders, 4th ed. (DSM-IV). Washington DC (USA), 1994.
34.
. Kähkönen M. Population cytogenetics of folate-sensitive fragile sites. Hum Genet 1988; 80:344-48.
35.
. Hungerford DA. Leukocytes cultured from small inocula of whole blood and the preparation of metaphase chromosomes by treatment with hypotonic KCl. Stain Technol 1965; 40:333-38.
36.
. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acid Res 1988; 16:1215.
37.
. Martin JP, Bell J. A pedigree of mental defect showing sex-linkage. J Neurol Psychiatry 1943; 6:154–57.
38.
. Magenis RE, Hecht F, Lovrien EW. Heritable Fragile Site on Chromosome 16: Probable Localization of Haptoglobin Locus in Man. Science 1970; 170: 85 - 87.
39.
. Sutherland GR. Fragile sites on human chromosomes: demonstration of their dependence on the type of tissue culture medium. Science 1977; 197: 265 - 66.
40.
. Maino DM, Maino JH, Maino SA. Mental retardation syndromes with associated ocular defects. J Am Optom Assoc 1990; 61:707-16.
41.
. Brown WT. Perspectives and molecular diagnosis of Fragile X syndrome. Clin Lab Med 1995; 15: 859-75.
42.
. Elango R, Verma IC. Fragile X syndrome among children with mental retardation. Indian J Pediatr 1996; 63:533-38.
43.
. Rousseau F, Rouillard P, Morel ML, et al. Prevalence of carriers of permutation-size alleles of the FMR1 gene and implications for the population genetics of Fragile X syndrome. Am J Hum Genet 1995; 57:1006-18.
44.
. Arinami T, Asano M, Kobayashi K, et al. The data on the CGG repeat at the fragile X site in the non-retarded Japanese population and family suggest the presence of a subgroup of normal alleles predisposing to mutation. Hum Genet 1993; 92:431-36.
45.
. Fu YH, Kuhl DPA, Pizzuti A, et al. Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox. Cell 1991; 67:1047-58.
46.
. Chen TA, Lu XF, Che PK, Ho Walter KK. Variation of the CGG repeat in the FMR-1 gene in normal and fragile X Chinese subjects. Ann Clin Biochem 1997; 34:517-20.

JCDR is now Monthly and more widely Indexed .
  • Emerging Sources Citation Index (Web of Science, thomsonreuters)
  • Index Copernicus ICV 2017: 134.54
  • Academic Search Complete Database
  • Directory of Open Access Journals (DOAJ)
  • Embase
  • EBSCOhost
  • Google Scholar
  • HINARI Access to Research in Health Programme
  • Indian Science Abstracts (ISA)
  • Journal seek Database
  • Google
  • Popline (reproductive health literature)
  • www.omnimedicalsearch.com