Biomedical subjects
Publications and source records attributed to I C Verma.
This editorial provides information on the first draft of the human genome, and examines the impact this will have on the practice of medicine, with special reference to India. It examines what is currently possible in India, what we should do in the immediate future, and what is in store in the long run. The future is indeed bright and India needs to expand the genetic services and research facilities.
A case of juvenile neuronal ceroid lipofuscinosis (JNCL) diagnosed on the basis of clinical features, electrophysiologic studies and skin electron microscopy is reported. JNCL was suspected on the basis of characteristic symptoms including progressive loss of vision, seizures, mental retardation and motor disabilities. Diagnosis was confirmed by neurophysiological and biopsy studies. The disease is caused by 23 different mutations in a gene recently isolated on chromosome 16 p11.2-12.1. Although universally fatal, characterisation of mutations can help in prenatal diagnosis in future pregnancies.
The challenges for childcare in the new millennium are outlined. The highly successful polio immunization strategy needs to be extended to other activities. The training programs developed for neonatal resuscitation, intensive care and care in oncology should be extended to other specialties. The journal will provide more fellowships and aid in the development of sub specialties in pediatrics. It will be published monthly and will also go online from January 2000.
Biotinidase deficiency is a well recognised treatable cause of a wide spectrum of progressive neurological symptoms. Recent reports have stressed the need to screen children with early onset of seizures, encephalopathy, neurodevelopmental delay, skin rash and alopecia. Enzyme estimation remains the conclusive test. We present a patient with biotinidase deficiency suspected on the above clinical grounds and diagnosed on the basis of metabolic acidosis, raised blood lactate, ketonuria and positive dinitrophenylhydrazine (DNPH) test and confirmed on urinary organic acid profile. Supplementation with biotin resulted in marked clinical improvement and normalisation of metabolic parameters. Thus the clinician should be alert to simple clinical pointers which aid in early diagnosis of these disorders.
Little is known about the heterozygous frequency of factor VIII gene markers in the Asian Indian population. The objective of this study was to establish the heterozygous frequency of polymorphic markers within and flanking the factor VIII gene in Indians and identify those most informative for carrier screening and prenatal diagnosis. Factor VIII gene polymorphism analysis at intragenic and extragenic sites was carried out by the polymerase chain reaction (PCR) method and Southern blot procedure. Sixty-three Asian Indian haemophiliacs and their families were screened. A control group of 150 women from nonhaemophilic families were screened for two markers, HindIII and BclI. Among the intragenic markers studied, the HindIII restriction fragment length polymorphism (RFLP) showed the highest heterozygous frequency (0.52) followed by the intron 13 (0.47) and intron 22 (0. 44) short tandem repeats (STRs). Among extragenic markers, TaqI had the highest heterozygous frequency (0.75) followed by BglII (0.54). The intron 22 inversion mutation was observed in eight (40%) of 20 severe cases. In the population studied the most diagnostic polymorphisms were the intragenic markers, intron 22 (70%) STR followed by the intron 13 (52%) STR and HindIII (52%) RFLP, and the TaqI (50%) extragenic marker. Application of HindIII, BclI and the intron 22 dinucleotide repeat combined were diagnostic in 87.2% of haemophilia A families studied.
We report the analysis of a beta-thalassaemia gene involving three bases in codons 4/5 and 6 (ACT CCT GAG-> ACA TCT TAG) in a confirmed carrier whose child had beta-thalassaemia major. The fragment of the gene carrying the mutation was detected by denaturing gradient gel electrophoresis (DGGE) using GC clamped primers, followed by direct sequencing. DGGE analysis indicated that one gene was the wild type (normal) while the sequence changes observed were all in the other gene causing beta-thalassaemia major in the child. This confirms a single case report from Lucknow (UP) and adds to the beta-thalassaemia mutations identified in the beta-globin gene in India and will help in the thalassaemia control programme.
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Autosomal recessive nonsyndromic sensorineural deafness segregating in a large consanguineous Indian family was mapped to chromosome 11p14-p15.1 defining a new locus, DFNB18. A maximum lod score of 4.4 at theta = 0 was obtained for the polymorphic micro-satellite marker D11S1888. Haplotype analysis localizes this gene between markers D11S1307 and D11S2368, which is approximately 1.6 cM and encompasses the region of Usher syndrome type 1C (USH1C). We postulate that DFNB18 and USH1C are allelic variants of the same gene.
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We present our experience with the amplification refractory mutation system (ARMS) for the prenatal diagnosis of beta-thalassaemia in 415 pregnancies of 360 women. Five mutations of the beta-thalassaemia gene common in Asian Indians accounted for 89.2 per cent and rare mutations for 7.2 per cent of all mutant chromosomes, while 3.3 per cent of chromosomes remained uncharacterized. Identical mutations were present in both parents in 43.2 per cent of cases, due to caste-based marriages in India. A confirmed diagnosis was given in 401 (98.3 per cent) cases, of which a complete diagnosis (whether the fetus was normal, a carrier, or homozygous) was possible in 391 (94.2 per cent) of the cases. In 15 couples, the mutation was identified in only one parent. In nine of these, the identified mutation was not present in the fetus, predicting normal/carrier status, while in five the identified mutation was present in the fetus, suggesting carrier/affected status. The abortion rate was 3.9 per cent. Pitfalls in diagnosis were failure of oligonucleotides to work, maternal contamination, and false paternity. The ARMS provides an inexpensive, robust and non-isotopic method for the prenatal diagnosis of beta-thalassaemia in India. Recommendations are outlined for establishing a prenatal diagnostic service in developing countries.
The utility of polymerase chain reaction (PCR) amplification of amelogenin gene as a reliable and rapid means of determination of sex chromosomes was tested in 20 patients of X-linked disorders (Duchenne muscular dystrophy, haemophilia and Wiscott-Aldrich and Hunter's syndromes), 12 of intersex (testicular feminization syndrome, male pseudohermaphrodites, true hermaphrodites) and 21 of congenital adrenal hyperplasia. Of these, 26 (49%) cases were for prenatal diagnosis of X-linked diseases and congenital adrenal hyperplasia (CAH). The presence of X and Y chromosomes was determined within 24 h of receiving the samples. The results were in conformity with cytogenetic studies in all instances. The analysis of amelogenin gene proved helpful in the diagnosis and management of these patients.
The usefulness of the clinical score based on Turner 5-trait scale prior to undertaking cytogenetic or molecular tests for the diagnosis of the fragile X(A) syndrome was evaluated. Mean clinical score in fragile X positive patients was significantly higher than in fragile X negative patients (7.06 +/- 1.85 vs 2.98 +/- 1.6, P < 0.0001). Of 1206 children with mental retardation 360 (29.8%) boys fulfilled defined clinical criteria to be screened for fragile X syndrome by chromosomal studies. Twenty three (6.38%) of them were found to be positive for fragile X syndrome using cytogenetic techniques. Molecular confirmation in 21 affected boys (two were lost to follow up) showed full mutation in 19 (5.27%). Two patients showed a normal 5.2 kb band on southern blot. This frequency (5.27%) of fragile X(A) patients among children with non-specific mental retardation is comparable to the results of studies in the West. Routine use of the clinical score, and the selection of patients with a score > or = 5 for diagnostic tests would reduce the laboratory load, especially in countries with limited facilities.
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We studied 160 cases of Duchenne muscular dystrophy (DMD) drawn from all parts of India, using multiplex PCR of 27 exons. Of these, 103 (64.4%) showed intragenic deletions. Most (69.7%) of the deletions involved exons 45-51. The phenotype of cases with deletion of single exons did not differ significantly from those with deletion of multiple exons. The distribution of deletions in studies from different countries was variable, but this was accounted for either by the small number of cases studied, or by fewer exons analyzed. It is concluded that there is likely to be no ethnic difference with respect to deletions in the DMD gene.
We have characterized the mutations in 1050 carriers of the beta-thalassemia gene and analyzed their regional distribution in India. The majority of beta-thalassemia carriers were migrants from Pakistan and their pattern of mutations differed from the rest. The frequency of the 619-bp deletion was 33.3% among the migrants from Pakistan, 8-17% in the northern states, and less than 5% in the other states. Among non-migrant subjects, the predominant mutation was IVS-I-5 (G-->C), varying from 85% in the southern states and 66-70% in the eastern states to 47-60% in the northern states. The mutation IVS-I-1 (G-->T) was observed at high frequency among the migrants from Pakistan (26.2%), but with very low/zero frequency in the other states. Mutations at codons 8/9 (+G) and codons 41/42 (-CTTT) were distributed in all regions of India with a frequency varying from 3% to 15%. Only eight of 12 published rare mutations were observed in subjects from different parts of India. Mutations of codon 5 (-CT) and codons 47/48 (+ATCT) were found exclusively in migrants from Pakistan, and mutation -88 (C-->T) was detected only in subjects from Punjab, Haryana, and Uttar Pradesh. Using the amplification refractory mutation system technique, mutations were successfully identified in 98.2% of subjects. Overall, 91.8% of the subjects had one of the five commonest mutations [IVS-I-5 (G-->C), 34.1%; 619-bp deletion, 21.0%; IVS-I-1 (G-->T) 15.8%; codons 8/9 (+G), 12.1%, and codons 41/42 (-CTTT), 8.7%], 5.9% of the subjects had a less common mutation, while 1.8% of the carriers remained uncharacterized. The application of this knowledge has helped to successfully establish a program of genetic counselling and prenatal diagnosis of beta-thalassemia in order to reduce the burden of this disease in India.
A study of 2570 tribals comprising 973 from Kerala, 696 from Madhya Pradesh and 901 from Orissa revealed the frequency of sickle cell gene to vary from 0.05 to 0.31 among different communities. High frequency of the gene (0.145 or more) was observed among Chettys, Kurmars and Kondhs, who also had a substantial number of homozygous sicklers. None of those with sickle cell disease (HbSS) were more than 39 yr in age as compared with 9.9-35.3 per cent among heterozygotes (HbAS). Mean foetal haemoglobin in those with sickle cell disease varied between 9.7 to 13.5 per cent, although ti showed a slight positive correlation with total haemoglobin only among the Kondhs. Painful crises were universally observed among all tribals with sickle cell disease, with jaundice being present in 57.5 per cent of cases. Some carriers of sickle cell gene also complained of painful crises. A health plan for identifying homozygotes in infancy with appropriate medical management is highly desirable.
We report the diagnosis of the newly identified mutation codon 47/48 insertion ATCT using allele specific primers by the PCR-based amplification refractory mutation system (ARMS). Using this method prenatal diagnosis was provided to five couples at risk for this mutation. The assay provides an easy, non-isotopic method for identifying this mutation in cases which remain uncharacterized after screening for the 5 'common' and 12 'rare' Indian mutations. It will be useful for screening and prenatal diagnosis in at risk couples and help in the thalassaemia control programme in India.
Chorionic villus sampling (CVS) offers rapid prenatal diagnosis of chromosomal disorders. We evaluated four methods for chromosomal analysis of chorionic villi (three based on direct preparations and one on long-term culture) in order to define a method which would provide good quality metaphases in sufficient numbers. The direct culturing method using synchronisation and dissociation with dispase was as good as long-term culturing. The optimal period of gestation for study was 10-12 wk.