Jayaprakash D. Karkera

CFC1 Mutations in Patients with Transposition of the Great Arteries and Double-Outlet Right Ventricle

Recent investigations identified heterozygous CFC1 mutations in subjects with heterotaxy syndrome, all of whom had congenital cardiac malformations, including malposition of the great arteries. We hypothesized that a subset of patients with similar types of congenital heart disease---namely, transposition of the great arteries and double-outlet right ventricle, in the absence of laterality defects---would also have CFC1 mutations. Our analysis of the CFC1 gene in patients with these cardiac disorders identified two disease-related mutations in 86 patients. The present study identifies the first autosomal single-gene defect for these cardiac malformations and indicates that some cases of transposition of the great arteries and double-outlet right ventricle can share a common genetic etiology with heterotaxy syndrome. In addition, these results demonstrate that the molecular pathway involving CFC1 plays a critical role in normal and abnormal cardiovascular development.

Molecular cytogenetic fingerprinting of esophageal squamous cell carcinoma by comparative genomic hybridization reveals a consistent pattern of chromosomal alterations.

Esophageal cancer is the third most prevalent gastrointestinal malignancy in the world. The tumor responds poorly to various therapeutic regimens and the genetic events underlying esophageal carcinogenesis are not well understood. To identify overall chromosomal aberrations in esophageal squamous cell carcinoma, we performed comparative genomic hybridization (CGH). All 17 tumor samples were found to exhibit multiple gains and losses involving different chromosomal regions. The frequency of chromosomal loss associated with this type of tumor was as follows: in 2q (100%), 3p (100%), 13q (100%), Xq (94%), 4 (82%), 5q (82%), 18q (76%), 9p (76%), 6q (70%), 12q (70%), 14q (65%), 11q (59%), and 1p (53%). Interstitial deletions on 1p, 3p, 5q, 6q, 11q, and 12q were detected also. Chromosomal gains were displayed by chromosomes and chromosome areas: 19 (100%), 20q (94%), 22 (94%), 16p (65%), 17 (59%), 12q (59%), 8q (53%), 9q (53%), and 3q (50%). Two sites showing apparent amplification were 11q (70%) and 5p15 (47%). To validate the CGH data, we isolated a BAC clone mapping to 18q12.1. This clone was used as a probe in interphase fluorescence in situ hybridization of tumor touch preparations and allelic loss was clearly revealed. This study represents the first whole‐genome analysis in esophageal squamous cell carcinoma for associated chromosomal aberrations that may be involved in either the genesis or progression of this malignancy. Genes Chromosomes Cancer 25:160–168, 1999.

Reduced NODAL Signaling Strength via Mutation of Several Pathway Members Including FOXH1 Is Linked to Human Heart Defects and Holoprosencephaly

Abnormalities of embryonic patterning are hypothesized to underlie many common congenital malformations in humans including congenital heart defects (CHDs), left-right disturbances (L-R) or laterality, and holoprosencephaly (HPE). Studies in model organisms suggest that Nodal-like factors provide instructions for key aspects of body axis and germ layer patterning; however, the complex genetics of pathogenic gene variant(s) in humans are poorly understood. Here we report our studies of FOXH1, CFC1, and SMAD2 and summarize our mutational analysis of three additional components in the human NODAL-signaling pathway: NODAL, GDF1, and TDGF1. We identify functionally abnormal gene products throughout the pathway that are clearly associated with CHD, laterality, and HPE. Abnormal gene products are most commonly detected in patients within a narrow spectrum of isolated conotruncal heart defects (minimum 5%-10% of subjects), and far less commonly in isolated laterality or HPE patients (approximately 1% for each). The difference in the mutation incidence between these groups is highly significant. We show that apparent gene dosage discrepancies between humans and model organisms can be reconciled by considering a broader combination of sequence variants. Our studies confirm that (1) the genetic vulnerabilities inferred from model organisms with defects in Nodal signaling are indeed analogous to humans; (2) the molecular analysis of an entire signaling pathway is more complete and robust than that of individual genes and presages future studies by whole-genome analysis; and (3) a functional genomics approach is essential to fully appreciate the complex genetic interactions necessary to produce these effects in humans.

Novel mutation in sonic hedgehog in non-syndromic colobomatous microphthalmia.

Ocular (uveoretinal) colobomas occur in one in 10,000 individuals and present a substantive cause of congenital poor vision. The genetic bases of most forms of uveoretinal coloboma are elusive; mutations in PAX2 are found in only a few cases of coloboma of the retina and optic nerve that occur with renal anomalies as part of the renal‐coloboma syndrome (MIM#120330; #167409). From experimental data that upstream expression of sonic hedgehog (SHH) controls Pax2 expression in mice and zebrafish, and from clinical experience that colobomas are observed frequently in patients with holoprosencephaly, we hypothesized that SHH could be a candidate for non‐syndromic ocular colobomas (NSOC). We identified a three‐generation family in which both a proband and his mother presented with iris and uveoretinal colobomas without optic nerve involvement. A novel 24 bp deletion in the gene SHH was identified in these affected family members, and cosegregated with the phenotype. This is the first report of the association of SHH mutations and uveoretinal coloboma.

Loss-of-Function Mutations in Growth Differentiation Factor-1 (GDF1) Are Associated with Congenital Heart Defects in Humans

Congenital heart defects (CHDs) are among the most common birth defects in humans (incidence 8-10 per 1,000 live births). Although their etiology is often poorly understood, most are considered to arise from multifactorial influences, including environmental and genetic components, as well as from less common syndromic forms. We hypothesized that disturbances in left-right patterning could contribute to the pathogenesis of selected cardiac defects by interfering with the extrinsic cues leading to the proper looping and vessel remodeling of the normally asymmetrically developed heart and vessels. Here, we show that heterozygous loss-of-function mutations in the human GDF1 gene contribute to cardiac defects ranging from tetralogy of Fallot to transposition of the great arteries and that decreased TGF- beta signaling provides a framework for understanding their pathogenesis. These findings implicate perturbations of the TGF- beta signaling pathway in the causation of a major subclass of human CHDs.

Cumulative ligand activity of NODAL mutations and modifiers are linked to human heart defects and holoprosencephaly

The cyclopic and laterality phenotypes in model organisms linked to disturbances in the generation or propagation of Nodal-like signals are potential examples of similar impairments resulting in birth defects in humans. However, the types of gene mutation(s) and their pathogenetic combinations in humans are poorly understood. Here we describe a mutational analysis of the human NODAL gene in a large panel of patients with phenotypes compatible with diminished NODAL ligand function. Significant reductions in the biological activity of NODAL alleles are detected among patients with congenital heart defects (CHD), laterality anomalies (e.g. left-right mis-specification phenotypes), and only rarely holoprosencephaly (HPE). While many of these NODAL variants are typical for family-specific mutations, we also report the presence of alleles with significantly reduced activity among common population variants. We propose that some of these common variants act as modifiers and contribute to the ultimate phenotypic outcome in these patients; furthermore, we draw parallels with strain-specific modifiers in model organisms to bolster this interpretation.

Missense Mutation of the MET Gene Detected in Human Glioma

Multiple mechanisms, such as gene mutations, amplifications, and rearrangements, as well as perturbed mitogen and receptor function, are likely to contribute to glioma formation. The MET (also known as c-met) proto-oncogene located at 7q31-34 has been shown to be amplified in human gliomas, and activating mutations within the tyrosine kinase domain of MET have been causally related to tumorigenesis in hereditary papillary renal cell carcinoma. To elucidate the role of MET gene in glioma formation, sporadic gliomas from 11 patients were examined for MET gene mutations and allelic duplications or deletions by polyermase chain reaction-single strand conformational polymorphism analysis and fluorescence in situ hybridization. Three of 11 sporadic gliomas showed a deletion of one copy of the MET gene, and a specific MET gene missense mutation in the remaining gene copy was detected in one of those tumors. The corresponding sequence in non-tumor DNA was normal in all cases. Three of 11 sporadic gliomas showed duplication of one copy of the MET gene, but none of them contained mutations. One tumor showed MET amplification without mutation. Three showed neither allelic change nor mutation. These data suggest that somatic MET gene mutation may play a role in the development of a subgroup of sporadic gliomas. However, MET mutations appear to be absent in the majority of sporadic gliomas.

Deletion of a consensus oestrogen response element half-site in the glucocorticoid receptor of human multiple myeloma

We have carried out molecular scanning of the glucocorticoid receptor (GR) of the glucocorticoid resistant multiple myeloma cell line U266. An amplified fragment from the 3′ untranslated region displayed an aberrant migration by PCR–single‐stranded conformational polymorphism (PCR‐SSCP) analysis. The mutant allele had a deletion of an 8 base pair sequence containing a half‐site of an oestrogen response element. This motif was found conserved in rat GR. This same allele lacked four As in an upstream region with 18 consecutive As in the normal allele. These mutations may affect mRNA stability or alter interactions with regulatory factors.

Systematic screening of chromosome 18 for loss of heterozygosity in esophageal squamous cell carcinoma.

Esophageal cancer ranks among the 10 most common cancers in the world, and is almost uniformly fatal. The genetic events leading to the development of esophageal carcinoma are not well established. To identify genomic regions involved in esophageal carcinogenesis, we performed a systematic screening for loss of heterozygosity (LOH) in 24 samples of squamous cell carcinomas, initially focusing the analysis on chromosome 18. Thirteen short tandem repeat markers spanning 18p and 18q were used. We found a broad peak of LOH spanning 18p11.2 and 18q21.1 with the most frequent LOH (72%) at D18S978 on 18q12.2, which coincides with a known fragile site FRA18A. This region is 4 cM proximal to known tumor suppressor genes and therefore suggests the possible existence of a yet undiscovered tumor suppressor gene.