Sequential occurrence of preneoplastic lesions and accumulation of loss of heterozygosity in patients with gallbladder stones suggest causal association with gallbladder cancer.

BACKGROUND: Causal association of gallbladder stones with gallbladder cancer (GBC) is not yet well established. OBJECTIVE: To study the frequency of occurrence of preneoplastic histological lesions and loss of heterozygosity (LOH) of tumor suppressor genes in patients with gallstones. METHODS: All consecutive patients with gallstones undergoing cholecystectomy from 2007-2011 were included prospectively. Histological examination of the gallbladder specimens was done for preneoplastic lesions. LOH at 8 loci, that is 3p12, 3p14.2, 5q21, 9p21, 9q, 13q, 17p13, and 18q for tumor suppressor genes (DUTT1, FHIT, APC, p16, FCMD, RB1, p53, and DCC genes) that are associated with GBC was tested from microdissected preneoplastic lesions using microsatellite markers. These LOH were also tested in 30 GBC specimens. RESULTS: Of the 350 gallbladder specimens from gallstone patients, hyperplasia was found in 32%, metaplasia in 47.8%, dysplasia in 15.7%, and carcinoma in situ in 0.6%. Hyperplasia, metaplasia, and dysplasia alone were found in 11.7%, 24.6%, and 1.4% of patients, respectively. A combination of hyperplasia and dysplasia, metaplasia and dysplasia, and hyperplasia, metaplasia, and dysplasia was found in 3.4%, 6.3%, and 4.3% of patients, respectively. LOH was present in 2.1% to 47.8% of all the preneoplastic lesions at different loci. Fractional allelic loss was significantly higher in those with dysplasia compared with other preneoplastic lesions (0.31 vs 0.22; P = 0.042). No preneoplastic lesion or LOH was found in normal gallbladders. CONCLUSIONS: Patients with gallstones had a high frequency of preneoplastic lesions and accumulation of LOH at various tumor suppressor genes, suggesting a possible causal association of gallstones with GBC.

Brachytelephalangic chondrodysplasia punctata: a case series to further delineate the phenotype.

Chondrodysplasia punctata (CDP) is a heterogenous group of skeletal dysplasias characterized by aberrant bone mineralization, manifesting radiologically as epiphyseal stippling. Among this group, brachytelephalangic dysplasia, a benign form of CDP (CDPX1), is probably under-reported. It is an X-linked recessive disorder and is characterized by a flat nasal tip, short columella and maxillary hypoplasia, involvement of terminal phalanges, and stippled chondrodystrophy. This paper presents a clinical series of 13 patients with brachytelephalangic dysplasia. These patients enrolled during 2002-2006 were re-evaluated and their dysmorphic features were compiled in a predesigned proforma. Skeletal survey, karyotype, cardiac evaluation, and ophthalmic evaluation were planned for all the cases. Out of 13 patients, 10 were males and three were females. All patients had flat facies, a depressed nasal bridge, a hypoplastic nose, a short philtrum, notched alae nasi, brachydactyly, and hypoplastic terminal phalanges. In addition, congenital heart disease, optic nerve hypoplasia, and developmental delay were found in a few patients. Radiography showed hypoplastic terminal phalanges, delayed bone age (1/13), epiphyseal stippling in carpal (3/13) and tarsal bones (2/13), sacral bone (1/13), and bullet-shaped lumbar vertebra (1/13). Cranial neuroimaging, thyroid profile, and karyotype carried out in a few were normal. The present paper discusses various clinical features and associated abnormalities in patients with brachytelephalangic dysplasia (CDPX1) to further delineate the phenotype. The presence of a similar phenotype in females suggests the possibility of another locus or manifestation of disease in heterozygous females. Arylsulfatase E gene analysis would further help in establishing the genotype-phenotype correlation.

Molecular genetic studies in Indian patients with megalencephalic leukoencephalopathy.

Mutations in the MLC1 gene cause megalencephalic leukoencephalopathy with subcortical cysts. We sought to identify mutations in the MLC1 gene, to evaluate the genotype-phenotype correlation, and to develop a strategy for diagnosing Indian patients with megalencephalic leukoencephalopathy. Forty patients were enrolled. We developed a rapid restriction fragment length polymorphism method to screen a common mutation, c.135_136insC. Rare and novel mutations were screened by conformation-sensitive gel electrophoresis, followed by sequencing. Three previously reported and two novel mutations were identified in 37 patients. The presence of the c.135_136insC mutation in 29 patients of the Agarwal community suggests a founder effect. The mutation c.959C>A was evident in four patients, and appears to be the second commonest mutation. Genotype could not predict phenotype. We recommend screening for the commonest mutation (c.135_136insC), followed by the next commonest mutation (c.959C>A), and then other rare mutations, using conformation-sensitive gel electrophoresis analysis or direct sequencing.

Noncoding mutations of HGF are associated with nonsyndromic hearing loss, DFNB39.

A gene causing autosomal-recessive, nonsyndromic hearing loss, DFNB39, was previously mapped to an 18 Mb interval on chromosome 7q11.22-q21.12. We mapped an additional 40 consanguineous families segregating nonsyndromic hearing loss to the DFNB39 locus and refined the obligate interval to 1.2 Mb. The coding regions of all genes in this interval were sequenced, and no missense, nonsense, or frameshift mutations were found. We sequenced the noncoding sequences of genes, as well as noncoding genes, and found three mutations clustered in intron 4 and exon 5 in the hepatocyte growth factor gene (HGF). Two intron 4 deletions occur in a highly conserved sequence that is part of the 3' untranslated region of a previously undescribed short isoform of HGF. The third mutation is a silent substitution, and we demonstrate that it affects splicing in vitro. HGF is involved in a wide variety of signaling pathways in many different tissues, yet these putative regulatory mutations cause a surprisingly specific phenotype, which is nonsydromic hearing loss. Two mouse models of Hgf dysregulation, one in which an Hgf transgene is ubiquitously overexpressed and the other a conditional knockout that deletes Hgf from a limited number of tissues, including the cochlea, result in deafness. Overexpression of HGF is associated with progressive degeneration of outer hair cells in the cochlea, whereas cochlear deletion of Hgf is associated with more general dysplasia.

Mutational spectrum of MYO15A: the large N-terminal extension of myosin XVA is required for hearing.

Human MYO15A is located on chromosome 17p11.2, has 66 exons and encodes unconventional myosin XVA. Recessive mutations of MYO15A are associated with profound, nonsyndromic hearing loss DFNB3 in humans, and deafness and circling behavior in shaker 2 mice. In the inner ear, this motor protein is necessary for the development of hair cell stereocilia, which are actin-filled projections on the apical surface and the site of mechanotransduction of sound. The longest isoform of myosin XVA has 3,530 amino acid residues. Two isoform classes of MYO15A are distinguished by the presence or absence of 1,203 residues preceding the motor domain encoded by alternatively-spliced exon 2. It is not known whether this large N-terminal extension of myosin XVA is functionally necessary for hearing. We ascertained approximately 600 consanguineous families segregating hereditary hearing loss as a recessive trait and found evidence of linkage of markers at the DFNB3 locus to hearing loss in 38 of these families ascertained in Pakistan (n=30), India (n=6), and Turkey (n=2). In this study, we describe 16 novel recessive mutations of MYO15A associated with severe to profound hearing loss segregating in 20 of these DFNB3-linked families. Importantly, two homozygous mutant alleles-c.3313G>T (p.E1105X) and c.3334delG (p.G1112fsX1124) of MYO15A-located in exon 2 are associated with severe to profound hearing loss segregating in two families. These data demonstrate that isoform 1, containing the large N-terminal extension, is also necessary for normal hearing.

In vivo assessment of toxicity and pharmacokinetics of methylglyoxal. Augmentation of the curative effect of methylglyoxal on cancer-bearing mice by ascorbic acid and creatine.

Previous in vivo studies from several laboratories had shown remarkable curative effect of methylglyoxal on cancer-bearing animals. In contrast, most of the recent in vitro studies have assigned a toxic role for methylglyoxal. The present study was initiated with the objective to resolve whether methylglyoxal is truly toxic in vivo and to reassess its therapeutic potential. Four species of animals, both rodent and non-rodent, were treated with different doses of methylglyoxal through oral, subcutaneous and intravenous routes. Acute (treatment for only 1 day) toxicity tests had been done with mouse and rat. These animals received 2, 1 and 0.3 g of methylglyoxal/kg of body weight in a day through oral, subcutaneous and intravenous routes respectively. Chronic (treatment for around a month) toxicity test had been done with mouse, rat, rabbit and dog. Mouse, rat and dog received 1, 0.3 and 0.1 g of methylglyoxal/kg of body weight in a day through oral, subcutaneous and intravenous routes respectively. Rabbit received 0.55, 0.3 and 0.1 g of methylglyoxal/kg of body weight in a day through oral, subcutaneous and intravenous routes respectively. It had been observed that methylglyoxal had no deleterious effect on the physical and behavioral pattern of the treated animals. Fertility and teratogenecity studies were done with rats that were subjected to chronic toxicity tests. It had been observed that these animals produced healthy litters indicating no damage of the reproductive systems as well as no deleterious effect on the offspring. Studies on several biochemical and hematological parameters of methylglyoxal-treated rats and dogs and histological studies of several organs of methylglyoxal-treated mouse were performed. These studies indicated that methylglyoxal had no apparent deleterious effect on some vital organs of these animals. A detailed pharmacokinetic study was done with mouse after oral administration of methylglyoxal. The effect of methylglyoxal alone and in combination with creatine and ascorbic acid on cancer-bearing animals had been investigated by measuring the increase in life span and tumor cell growth inhibition. The results indicated that anticancer effect of methylglyoxal was significantly augmented by ascorbic acid and further augmented by ascorbic acid and creatine. Nearly 80% of the animals treated with methylglyoxal plus ascorbic acid plus creatine were completely cured and devoid of any malignant cells within the peritoneal cavity.

DFNB3, spectrum of MYO15A recessive mutant alleles and an emerging genotype-phenotype correlation.

We have now identified seven MYO15A mutations that cause congenital profound neurosensory hearing loss and a possible hypomorphic allele of MYO15A associated with moderately-severe hearing loss in 1 of 8 SMS patients. Because myosin XVA is encoded by 66 exons, screening for mutations in hearing-impaired individuals is expensive and labor-intensive in comparison to a screen for mutations in GJB2 (Cx26), for example, which has only a single protein coding exon. Among consanguineous families segregating profound, congenital hearing loss from Pakistan, approximately 10% are consistent with linkage to DFNB3 (11 of 112 DFNB families). In one-half of these DFNB3 families, we found a homozygous mutation in 1 of the 66 exons of MYO15A [25]. This suggests that mutations of MYO15A are responsible for at least 5% of recessively inherited, profound hearing loss in Pakistan. However, without the benefit of a pre-screen for linkage to DFNB3, it will be a challenge to determine the extent to which mutations of MYO15A contribute to hereditary hearing loss among isolated cases and small families in other populations.

Nonsyndromic recessive deafness DFNB18 and Usher syndrome type IC are allelic mutations of USHIC.

Human chromosome 11 harbors two Usher type I loci, USHIB and USHIC, which encode myosin VIIA and harmonin, respectively. The USHIC locus overlaps the reported critical interval for nonsyndromic deafness locus DFNB18. We found an IVS12+5G-->C mutation in the USHIC gene, which is associated with nonsyndromic recessive deafness ( DFNB18) segregating in the original family, S-11/12. No other disease-associated mutation was found in the other 27 exons or in the intron-exon boundaries, and the IVS12+5G-->C mutation was not present in 200 representative unaffected individuals ascertained from the same area of India. An exon-trapping assay with a construct harboring IVS12+5G-->C generated wildtype spliced mRNA having exons 11 and 12 and mRNA that skipped exon 12. We conclude that mutations of USHIC can cause both Usher syndrome type IC and nonsyndromic recessive deafness DFNB18.