Conventional vs virtual autopsy with postmortem MRI in phenotypic characterization of stillbirths and fetal malformations.

OBJECTIVE: To compare virtual autopsy using postmortem magnetic resonance imaging (MRI) with conventional autopsy with respect to phenotypic characterization of stillbirths and malformed fetuses, and acceptability to parents. METHODS: This was a prospective diagnostic evaluation study, conducted from June 2013 to June 2015, including stillbirths and pregnancies terminated owing to fetal malformation at ≥ 20 weeks' gestation, for which parental consent to both conventional autopsy and postmortem MRI was obtained. Cases of maternal and obstetric cause of fetal demise were excluded. Whole-body postmortem MRI (at 1.5 T) was performed prior to conventional autopsy. Taking conventional autopsy as the diagnostic gold standard, postmortem MRI findings alone, or in conjunction with other minimally invasive prenatal and postmortem investigations, were assessed and compared for diagnostic accuracy. RESULTS: Parental consent for both conventional autopsy and postmortem MRI was obtained in 52 cases of which 43 were included in the analysis. In 35 (81.4%) cases, the final diagnosis based on virtual autopsy with postmortem MRI was in agreement with that of conventional autopsy. With conventional autopsy as the reference standard, sensitivity, specificity, positive and negative predictive values of postmortem MRI were, respectively: 77.7%, 99.8%, 97.4% and 98.0% for whole-body assessment; 93.1%, 99.0%, 87.1% and 99.5% for the nervous system; 61.0%, 100.0%, 100.0% and 96.7% for the cardiovascular system; 91.1%, 100.0%, 100.0% and 98.0% for the pulmonary system; 80.6%, 99.8%, 96.7% and 98.7% for the abdomen; 96.2%, 99.7%, 96.2% and 99.7% for the renal system; and 66.7%, 100.0%, 100.0% and 97.2% for the musculoskeletal system. Virtual autopsy was acceptable to 96.8% of families as compared with conventional autopsy to 82.5%. CONCLUSIONS: Virtual autopsy using postmortem MRI and other minimally invasive investigations can be an acceptable alternative to conventional autopsy when the latter is refused by the parents. Postmortem MRI is more acceptable to parents and can provide additional diagnostic information on brain and spinal cord malformations. Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd.

Nasopharyngeal teratoma, congenital diaphragmatic hernia and Dandy-Walker malformation - a yet uncharacterized syndrome.

An association of congenital diaphragmatic hernia, dandy walker malformation and nasopharyngeal teratoma is very rare. Here, we report a fourth case with this association where chromosomal microarray and whole exome sequencing (WES) was performed to understand the underlying genetic basis. Findings of few variants especially a novel variation in HIRA provided some insights. An association of congenital diaphragmatic hernia, dandy walker malformation and nasopharyngeal teratoma is very rare. Here, we report a fourth case with this association where chromosomal microarray and whole exome sequencing (WES) was performed to understand the underlying genetic basis. Findings of few variants especially a novel variation in HIRA provided some insights.

Mutation spectrum in BBS genes guided by homozygosity mapping in an Indian cohort.

Bardet-Biedl syndrome (BBS), a ciliopathy disorder with pleiotropic effect manifests primarily as retinal degeneration along with renal insufficiency, polydactyly and obesity. In this study, we have performed homozygosity mapping using NspI 250K affymetrix gene chip followed by mutation screening of the candidate genes located in the homozygous blocks. These regions are prioritized based on the block length and candidature of the genes in BBS and other ciliopathies. Gene alterations in known BBS (22) and other ciliopathy genes such as ALMS1 (2) were seen in 24 of 30 families (80%). Mutations in BBS3 gene, inclusive of a novel recurrent mutation (p.I91T) accounted for 18% of the identified variations. Disease associated polymorphisms p.S70N (BBS2), rs1545 and rs1547 (BBS6) were also observed. This is the first study in Indian BBS patients and homozygosity mapping has proved to be an effective tool in prioritizing the candidate genes in consanguineous pedigrees. The study reveals a different mutation profile in the ciliopathy genes in Indian population and implication of novel loci/genes in 20% of the study group.

Threshold levels of 25-hydroxyvitamin D and parathyroid hormone for impaired bone health in children with congenital ichthyosis and type IV and V skin.

BACKGROUND: Patients with congenital ichthyosis, especially those with darker skin types, are at increased risk of developing vitamin D deficiency and rickets. The relationships between 25-hydroxyvitamin D [25(OH)D], parathyroid hormone (PTH) and bone health have not been studied previously, in ichthyosis. OBJECTIVES: To determine the threshold levels of 25(OH)D and PTH for impaired bone health in children with congenital ichthyosis. METHODS: In this cross-sectional study, 119 children with ichthyosis and 168 controls were recruited. Serum 25(OH)D, PTH, calcium, phosphate and alkaline phosphatase (ALP) were measured. Radiological screening for rickets was carried out only in children with ichthyosis. RESULTS: Forty-seven children with ichthyosis had either clinical or radiological evidence of rickets. The correlation between serum 25(OH)D and PTH showed that a serum level of 25(OH)D 8 ng mL(-1) was associated with a significant increase in PTH. The correlation between PTH and ALP showed that a serum PTH level of 75 pg mL(-1) was associated with a significant increase in ALP levels. Of the different clinical phenotypes of ichthyosis, both autosomal recessive congenital ichthyosis (ARCI) and epidermolytic ichthyosis (EI) were found to have significantly increased PTH, ALP and radiological rickets scores compared with common ichthyosis. CONCLUSIONS: Serum levels of 25(OH)D ≤ 8 ng mL(-1) and PTH ≥ 75 pg mL(-1) significantly increases the risk for development of rickets [odds ratio (OR) 2·8; 95% confidence interval (CI) 1·05-7·40; P = 0·04] in ichthyosis. Among the different types, patients with ARCI (OR 4·83; 95% CI 1·74-13·45; P < 0·01) and EI (OR 5·71; 95% CI 1·74-18·79; P < 0·01) are at an increased risk of developing rickets.

Molecular analyses of novel ASAH1 mutations causing Farber lipogranulomatosis: analyses of exonic splicing enhancer inactivating mutation.

Farber lipogranulomatosis is a rare autosomal recessive lysosomal storage disorder caused by mutations in the ASAH1 gene. In the largest ever study, we identified and characterized ASAH1 mutations from 11 independent Farber disease (FD) families. A total of 13 different mutations were identified including 1 splice, 1 polypyrimidine tract (PPT) deletion and 11 missense mutations. Eleven mutations were exclusive to the Indian population. The IVS6+4A>G splice and IVS5-16delTTTTC PPT deletion mutations resulted in skipping of exon 6 precluding thereby the region responsible for cleavage of enzyme precursor. A missense mutation (p.V198A) resulted in skipping of exon 8 due to inactivation of an exonic splicing enhancer (ESE) element. This is the first report of mutations affecting PPT and ESE in the ASAH1 gene resulting in FD.

Vitamin D deficiency and rickets in children and adolescents with ichthyosiform erythroderma in type IV and V skin.

BACKGROUND: Ichthyosiform erythroderma due to keratinizing disorders may suppress cutaneous vitamin D synthesis, leading to vitamin D deficiency and rickets. OBJECTIVES: To determine the prevalence of vitamin D deficiency and rickets in children and adolescents with congenital ichthyosis and other keratinizing disorders with erythroderma and scaling. PATIENTS AND METHODS: In this cross-sectional study, 45 children and adolescents with ichthyosiform erythroderma due to keratinizing disorders, and 66 controls (group 1: age and sex matched, with skin diseases other than keratinizing disorders; group 2: age and sex matched, healthy volunteers) were included. Evidence of rickets was determined clinically (physical examination and radiographs) and biochemically {serum calcium, phosphorus, alkaline phosphatase, 25-hydroxy vitamin D [25(OH)D] and parathyroid hormone (PTH)}. RESULTS: All patients in the disease group had clinical, radiological or biochemical evidence of rickets [25(OH)D<20ngmL(-1) ], and analysis was done for all subjects with the available biochemical reports. The mean serum 25(OH)D levels of the disease group was 8·38±5·23ngmL(-1) and was significantly lower than in control group 1 (11·1±5·8ngmL(-1) ) (P<0·01) and control group 2 (13·5±6·9ngmL(-1) ) (P<0·001). The prevalence of vitamin D deficiency [25(OH)D<20ngmL(-1) ] was significantly higher in the disease group (n=38 of 39, 97·4%) than in control group 2 (n=12, 70·6%) (P<0·01), and total controls (n=56, 84·8%) (P=0·04). The frequency of hyperparathyroidism (PTH>65pgmL(-1) ) was also significantly higher in the disease group than in controls (P<0·01). CONCLUSIONS: Children and adolescents with various forms of ichthyosiform erythroderma, especially those with pigmented skin (types IV-VI), are at increased risk of developing vitamin D deficiency and clinical rickets.

A founder ectodysplasin A receptor (EDAR) mutation results in a high frequency of the autosomal recessive form of hypohidrotic ectodermal dysplasia in India.

BACKGROUND: Hypohidrotic/anhidrotic ectodermal dysplasia (HED) is a rare Mendelian disorder affecting ectodermal tissues. The disease is primarily caused by inactivation of any one of three genes, namely ectodysplasin A1 (EDA-A1), which encodes a ligand belonging to the tumour necrosis factor (TNF) superfamily; ectodysplasin A receptor (EDAR), encoding the EDA-A1 receptor and ectodysplasin A receptor-associated death domain (EDARADD), encoding an adaptor protein. X-linked recessive (EDA-A1), the predominant form of HED, as well as autosomal recessive and dominant (EDAR and EDARADD) inheritance patterns have been identified in affected families. OBJECTIVES: To determine the common genes causing HED in India. METHODS: We performed mutation analysis on 26 HED families from India (including 30 patients). In addition, we carried out sequence and structural analysis of missense/nonsense and insertion/deletion mutations. RESULTS: Among the 26 families analysed, disease-causing EDAR mutations were identified in 12 (46%) while EDA-A1 mutations were detected in 11 (42%). Four novel mutations in EDAR and five in EDA-A1 were identified. More importantly, a possible founder EDAR mutation, namely c.1144G>A, was identified in five independent families, thus accounting for about one-fifth of affected families in whom mutation was detected. A majority of EDA-A1 mutations localized to the TNF-like domain while the location of EDAR mutations was more widespread. CONCLUSIONS: This is the first report of a founder EDAR mutation and of a significantly high frequency of autosomal recessive HED.

Rhizomelic chondrodysplasia punctata type 1: report of mutations in 3 children from India.

Rhizomelic chondrodysplasia punctata is a rare autosomal recessive disorder characterized by stippled epiphyses and rhizomelic shortening of the long bones. We report 3 subjects of rhizomelic chondrodysplasia punctata from India and the PEX7 mutations identified in them. The common PEX7-L292X allele, whose high frequency is due to a founder effect in the northern European Caucasian population, was not identified in these patients. Instead, 2 novel alleles are described, including 64_65delGC, which was present on a single PEX7 haplotype and could represent a common allele in the Indian population.

Cystic fibrosis in India.

Cystic fibrosis (CF) was considered to be non-existent in Indian subcontinent. Reports in last one decade have suggested that cystic fibrosis occurs in India but its precise magnitude is not known. Studies on migrant Indian population in United States and United Kingdom estimate frequency of CF as 1:10,000 to 1:40,000. The clinical features are similar to that reported in Caucasian population. CF in Indian children is usually diagnosed late and in advanced stage. Children are more malnourished and may have clinically evident deficiency of fat soluble vitamins. The frequency of clubbing, colonization with Pseudomonas, and laboratory evidence of pseudo-Bartter syndrome is relatively more at the time of diagnosis. Diagnostic facilities in form of sweat chloride estimation and genetic studies are not available readily. Mutation profile is different. The frequency of common mutation F508del in Indian children is between 19% and 34%. Other mutations are heterogeneous. Management of CF in India is difficult due to less number of trained manpower, limited availability, and high cost of pharmacologic agents. The determinants of early death include: severe malnutrition and colonization with Pseudomonas at the time of diagnosis, more than four episodes of lower respiratory infection per year and age of onset of symptoms before 2 months of age. To conclude, CF does occur in India; however, precise magnitude of problem is not known. There is need to create awareness amongst pediatricians, developing diagnostic facilities, and management protocols based on locally available resources.

Mutations of human TMHS cause recessively inherited non-syndromic hearing loss.

BACKGROUND: Approximately half the cases of prelingual hearing loss are caused by genetic factors. Identification of genes causing deafness is a crucial first step in understanding the normal function of these genes in the auditory system. Recently, a mutant allele of Tmhs was reported to be associated with deafness and circling behaviour in the hurry-scurry mouse. Tmhs encodes a predicted tetraspan protein of unknown function, which is expressed in inner ear hair cells. The human homologue of Tmhs is located on chromosome 6p. OBJECTIVE: To determine the cause of deafness in four consanguineous families segregating recessive deafness linked to markers on chromosome 6p21.1-p22.3 defining a novel DFNB locus. RESULTS: A novel locus for non-syndromic deafness DFNB67 was mapped in an interval of approximately 28.51 cM on human chromosome 6p21.1-p22.3. DNA sequence analysis of TMHS revealed a homozygous frameshift mutation (246delC) and a missense mutation (Y127C) in affected individuals of two families segregating non-syndromic deafness, one of which showed significant evidence of linkage to markers in the DFNB67 interval. The localisation of mTMHS in developing mouse inner ear hair cells was refined and found to be expressed briefly from E16.5 to P3. CONCLUSIONS: These findings establish the importance of TMHS for normal sound transduction in humans.

Arsenic induced apoptosis in rat liver following repeated 60 days exposure.

BACKGROUND: Accumulation of the wide spread environmental toxin arsenic in liver results in hepatotoxcity. Exposure to arsenite and other arsenicals has been previously shown to induce apoptosis in certain tumor cell lines at low (1-3 microM) concentration. AIM: The present study was focused to elucidate the role of free radicals in arsenic toxicity and to investigate the nature of in vivo sodium arsenite induced cell death in liver. METHODS: Male wistar rats were exposed to arsenite at three different doses of 0.05, 2.5 and 5mg/l for 60 days. Oxidative stress in liver was measured by estimating pro-oxidant and antioxidant activity in liver. Histopathological examination of liver was carried out by light and transmission electron microscopy. Analysis of DNA fragmentation by gel electrophoresis was used to identify apoptosis after the exposure. Terminal deoxy-nucleotidyl transferase mediated dUTP Nick end-labeling (TUNEL) assay was used to qualify and quantify apoptosis. RESULTS: A significant increase in cytochrome-P450 and lipid peroxidation accompanied with a significant alteration in the activity of many of the antioxidants was observed, all suggestive of arsenic induced oxidative stress. Histopathological examination under light and transmission electron microscope suggested a combination of ongoing necrosis and apoptosis. DNA-TUNEL showed an increase in apoptotic cells in liver. Agarose gel electrophoresis of DNA of hepatocytes resulted in a characteristic ladder pattern. CONCLUSION: Chronic arsenic administration induces a specific pattern of apoptosis called post-mitotic apoptosis.

Origins and frequencies of SLC26A4 (PDS) mutations in east and south Asians: global implications for the epidemiology of deafness.

Recessive mutations of SLC26A4 (PDS) are a common cause of Pendred syndrome and non-syndromic deafness in western populations. Although south and east Asia contain nearly one half of the global population, the origins and frequencies of SLC26A4 mutations in these regions are unknown. We PCR amplified and sequenced seven exons of SLC26A4 to detect selected mutations in 274 deaf probands from Korea, China, and Mongolia. A total of nine different mutations of SLC26A4 were detected among 15 (5.5%) of the 274 probands. Five mutations were novel and the other four had seldom, if ever, been identified outside east Asia. To identify mutations in south Asians, 212 Pakistani and 106 Indian families with three or more affected offspring of consanguineous matings were analysed for cosegregation of recessive deafness with short tandem repeat markers linked to SLC26A4. All 21 SLC26A4 exons were PCR amplified and sequenced in families segregating SLC26A4 linked deafness. Eleven mutant alleles of SLC26A4 were identified among 17 (5.4%) of the 318 families, and all 11 alleles were novel. SLC26A4 linked haplotypes on chromosomes with recurrent mutations were consistent with founder effects. Our observation of a diverse allelic series unique to each ethnic group indicates that mutational events at SLC26A4 are common and account for approximately 5% of recessive deafness in south Asians and other populations.

Cord blood analysis for prenatal diagnosis of thalassemia major and hemophilia A.

Beta thalassemia and Hemophilia A are common genetic disorders for which prenatal diagnosis (PND) is an accepted option. Our aim was to evaluate cord blood analysis as a method for PND of these disorders. Cord blood samples at 18-26 weeks gestation from nine mothers with previous thalassemia major child and five families with previous hemophilia A were studied. In the former; HbF, HbA2 and HbF were determined by high performance liquid chromatography (HPLC) and in latter; Factor VIII and IX assays were done by one stage method. In HPLC studies for thalassemia, three out of nine fetuses were affected, five were carriers and one was normal. In hemophilia PND samples, 2 out of five fetuses were affected. Thus, HPLC and factor VIII assay in cord blood are feasible alternatives for PND in Beta thalassemia and hemophilia A respectively, especially when DNA analysis is uninformative or there are financial constraints.

Blepharophimosis, telecanthus, microstomia, and unusual ear anomaly (Simosa syndrome) in an infant.

We report on a 1-month-old infant with blepharophimosis, telecanthus, ear anomalies, and microstomia. He also had a long philtrum and a narrow, high-arched palate. This case is very similar to the cases reported by Simosa et al. [1989: Am J Med Genet 32:184-186]. In addition, our patient had tongue tie and camptodactyly of fingers.

Poland anomaly with unusual associated anomalies: case report of an apparent disorganized defect.

We report on a 2 1/2-year-old boy with absence of clavicular head of pectoralis major on the left side, ipsilateral upper limb anomalies, and anomalies of the lower limbs such as popliteal webbing, median cleft of right foot, bifid left hallux, syndactyly of toes, and toenail hypoplasia. Other anomalies included undescended testis, hairy nevus in the lumbosacral region, and a pedunculated finger-like tag on the right thigh. The pathogenesis of these associated anomalies cannot be explained on the basis of compromised local blood supply alone. A possible link with the disorganization mutation is discussed.

Karyotyping of at risk fetuses by cordocentesis in advanced gestation.

Fetal blood obtained by cordocentesis was cultured to obtain rapid karyotypes of fetuses at risk during the late second trimester. Ninety nine fetal blood samples were studied for chromosomal abnormalities. The commonest indications for the procedure were abnormalities detected on ultrasonography (47.7%), and previous child with Down syndrome. Analysis of the 67 successful cultures showed four (5.9%) karyotypic abnormalities. The technique proved helpful in the obstetrical management of at risk fetuses.

Delta F 508 molecular mutation in Indian children with cystic fibrosis.

A preliminary report of 13 Indian children with cystic fibrosis who were screened for the commonest mutation (delta F 508) is presented. Six (46%) patients were homozygous for delta F 508, while two patients were compound heterozygotes. Thus 14 (53.8%) of 26 mutant chromosomes had delta F 508 mutation. These findings confirm that cystic fibrosis occurs in India and all children with persistent respiratory problems and/or malabsorption should be screened for this disease.

Molecular characterization of mutations in Indian children with congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency.

Congenital adrenal hyperplasia (CAH) is the commonest cause of female pseudohermaphroditism. It is most often due to steroid 21-hydroxylase deficiency resulting from mutations in the CYP21 gene. This study was conducted to characterize mutations in the CYP21 gene, determine their frequency and correlate genotype with phenotype in Indian children with CAH. Twenty-eight patients with salt-wasting (SW) or simple-virilizing (SV) forms of the disease as well as parents and siblings were studied. Allele specific PCR was carried out and rapid characterization of six mutations was achieved in 23 patients. Twelve patients were homozygous for the mutations and 11 were heterozygous, of whom eight were compound heterozygotes and three were hemizygotes; no mutation was found in five patients. The homozygosity of the mutations was found to be high in our population. The most common mutation was Ile173Asn (31.8%), followed by intron2 splice (27.2%), Gln319stop (22.7%), gene deletion (15.9%) and Pro31Leu (2.2%). Genotype-phenotype correlations showed that the most frequent mutations in the SW group were intron2 splice and Gln319stop mutations (33.3% each) and Ile173Asn (71.4%) in the SV group.

Diagnosis and management of congenital adrenal hyperplasia: clinical, molecular and prenatal aspects.

Congenital adrenal hyperplasia (CAH) is the most common cause of female pseudohermaphroditism in Indian children. It is caused by enzymatic defects in the steroidogenic pathway of the adrenal glands and is characterized by impaired cortisol and aldosterone synthesis and overproduction of androgens. The disease usually presents with life-threatening problems and virilization, with long term physical and psychological effects. The clinical and laboratory diagnoses play an important role in deciding the course of treatment, which continues lifelong. To ensure proper growth and development of the patient, optimized disease management and treatment with steroids is required. Often the patient also requires surgical correction. Recent developments in molecular genetics have greatly helped in understanding the pathogenesis of the disease. The gene encoding for steroid 21-hydroxylase, CYP21, is located on the short arm of chromosome 6 in the HLA region and is amplified for genetic diagnosis. Rapid characterization of point mutations is possible using the allele-specific polymerase chain reaction technique in affected children. Counselling, prenatal diagnosis and treatment are recommended in all pregnant women with a positive family history to reduce or eliminate the effects in affected foetuses. This spares the female newborn the consequences of genital ambiguity and problems of gender identity.