Laura Kasch

Cloning of the gamma-aminobutyric acid (GABA) rho 1 cDNA: a GABA receptor subunit highly expressed in the retina.

Type A gamma-aminobutyric acid (GABAA) receptors are a family of ligand-gated chloride channels that are the major inhibitory neurotransmitter receptors in the nervous system. Molecular cloning has revealed diversity in the subunits that compose this heterooligomeric receptor, but each previously elucidated subunit displays amino acid similarity in conserved structural elements. We have used these highly conserved regions to identify additional members of this family by using the polymerase chain reaction (PCR). One PCR product was used to isolate a full-length cDNA from a human retina cDNA library. The mature protein predicted from this cDNA sequence in 458 amino acids long and displays between 30 and 38% amino acid similarity to the previously identified GABAA subunits. This gene is expressed primarily in the retina but transcripts are also detected in the brain, lung, and thymus. Injection of Xenopus oocytes with RNA transcribed in vitro produces a GABA-responsive chloride conductance and expression of the cDNA in COS cells yields GABA-displaceable muscimol binding. These features are consistent with our identification of a GABA subunit, GABA rho 1, with prominent retinal expression that increases the diversity and tissue specificity of this ligand-gated ion-channel receptor family.

Genetic and environmental risk factors for sagittal craniosynostosis.

The authors investigated whether genetic and environmental factors influence risk for sagittal craniosynostosis. Cases were ascertained from craniofacial clinics in the Baltimore–Washington metropolitan region. Controls were recruited from the Johns Hopkins newborn nursery and a large pediatric practice in Baltimore County. Forty-two probands with isolated, nonsyndromic sagittal craniosynostosis born in the mid-Atlantic region were included in this analysis. Controls are infants born in Maryland without any known birth defects (n = 182). Odds ratios (OR) and corresponding 95% confidence intervals (CI) were calculated. Cases were genotyped at several loci implicated in malformation syndromes including craniosynostosis. There were no elevated risks for craniosynostosis related to maternal or paternal smoking or maternal vitamin usage. Case mothers consumed less alcohol (OR = 0.38, 95% CI = 0.17–0.85) and had less education than control mothers (P < 0.001). All cases that were sequenced were negative for mutations at the following genes:FGFR1 exon IIIa 755C->G, FGFR2 (exons IIIa and IIIc,), FGFR3 exon IIIa, and TWIST exon 1. These findings suggest that whereas TWIST and the FGFR genes are important for syndromic craniosynostosis, they are unlikely to be involved in isolated sagittal craniosynostosis. Parental education and alcohol consumption were associated with sagittal craniosynostosis in this study.

Inherited surfactant deficiency caused by uniparental disomy of rare mutations in the surfactant protein-B and ATP binding cassette, subfamily a, member 3 genes.

OBJECTIVE To characterize inheritance of homozygous, rare, recessive loss-of-function mutations in surfactant protein-B (SFTPB) or ATP binding cassette, subfamily A, member 3 (ABCA3) genes in newborns with lethal respiratory failure. STUDY DESIGN We resequenced genes from parents whose infants were homozygous for mutations in SFTPB or ABCA3. For infants with only 1 heterozygous parent, we performed microsatellite analysis for chromosomes 2 (SFTPB) and 16 (ABCA3). RESULTS We identified 1 infant homozygous for the g.1549C > GAA mutation (121ins2) in SFTPB for whom only the mother was heterozygous and 3 infants homozygous for mutations in ABCA3 (p.K914R, p.P147L, and c.806_7insGCT) for whom only the fathers were heterozygous. For the SP-B-deficient infant, microsatellite markers confirmed maternal heterodisomy with segmental isodisomy. Microsatellite analysis confirmed paternal isodisomy for the 3 ABCA3-deficient infants. Two ABCA3-deficient infants underwent lung transplantation at 3 and 5 months of age, respectively, and 2 infants died. None exhibited any nonpulmonary phenotype. CONCLUSIONS Uniparental disomy should be suspected in infants with rare homozygous mutations in SFTPB or ABCA3. Confirmation of parental carrier status is important to provide recurrence risk and to monitor expression of other phenotypes that may emerge through reduction to homozygosity of recessive alleles.

A paternally derived inverted duplication of 7q with evidence of a telomeric deletion

We report on a de novo constitutional rearrangement involving the long arm of chromosome 7 in a second trimester fetus with the karyotype of 46,XX, inv dup del (7)(pter-q36::q36-q21.2:) pat. Both a large duplication (q21.2-q36) and a small deletion (within q36) were confirmed by FISH studies. DNA analysis on the family showed that the abnormal chromosome was derived from a single paternal homolog. A mechanism is proposed in light of this finding. The phenotype at autopsy was consistent with reported cases of similar duplications in chromosome 7 in that hydrocephalus, a depressed nasal bridge, low set ears, microretrognathia and a short neck were present.

The use of polymerase chain reaction to determine fetal RhD status

OBJECTIVE Our purpose was (1) to establish the accuracy of a deoxyribonucleic acid amplification method in determination of RhD status in adult blood samples, including weak D variants (previously referred to as Du) and a D mosaic, and (2) to apply the method to determine fetal RhD status in alloimmunized pregnancies. STUDY DESIGN Twenty-five adult blood samples, including five weak D variants and one D mosaic, were analyzed with a polymerase chain reaction to determine RhD type. The method was then applied to amniotic fluid samples obtained by amniocentesis from three RhD-negative women with known RhD sensitization. RESULTS RhD type determined by polymerase chain reaction for all adult blood samples agreed with serologic typing results. All weak D variants and the D mosaic gave results consistent with RhD positivity. Fetal RhD status was determined in each of the three alloimmunized pregnancies, and obstetric management decisions were made on the basis of these results. CONCLUSIONS This polymerase chain reaction method allows rapid and accurate determinations of fetal RhD status by amniocentesis. Fetal blood sampling or serial amniocenteses may be avoided when the fetus is RhD negative, and plans for surveillance and intervention can be confidently made if the fetus is RhD positive. However, before the widespread use of this assay, its sensitivity and specificity must be established. Because weak D variants and a D mosaic demonstrated RhD-positive status by polymerase chain reaction, the method described is applicable to these RhD variants.

Identification of an HMGB3 Frameshift Mutation in a Family With an X-linked Colobomatous Microphthalmia Syndrome Using Whole-Genome and X-Exome Sequencing

IMPORTANCE Microphthalmias are rare disorders whose genetic bases are not fully understood. HMGB3 is a new candidate gene for X-linked forms of this disease. OBJECTIVE To identify the causative gene in a pedigree with an X-linked colobomatous microphthalmos phenotype. DESIGN, SETTING, AND PARTICIPANTS Whole-genome sequencing and chromosome X-exome-targeted sequencing were performed at the High Throughput Sequencing Laboratory of the Genetic Resources Core Facility at the Johns Hopkins University School of Medicine on the DNA of the male proband and informatically filtered to identify rare variants. Polymerase chain reaction and Sanger sequencing were used to confirm the variant in the proband and the carrier status of his mother. Thirteen unrelated male patients with a similar phenotype were also screened. MAIN OUTCOMES AND MEASURES Whole-genome and X-exome sequencing to identify a frameshift variant in HMGB3. RESULTS A 2-base pair frameshift insertion (c.477_478insTA, coding for p.Lys161Ilefs*54) in the HGMB3 gene was found in the proband and his carrier mother but not in the unrelated patients. The mutation, confirmed by 3 orthogonal methods, alters an evolutionarily conserved region of the HMGB3 protein from a negatively charged polyglutamic acid tract to a positively charged arginine-rich motif that is likely to interfere with normal protein function. CONCLUSIONS AND RELEVANCE In this family, microphthalmia, microcephaly, intellectual disability, and short stature are associated with a mutation on the X chromosome in the HMGB3 gene. HMGB3 should be considered when performing genetic studies of patients with similar phenotypes.

Physical map of the chromosome 6q22 region containing the oculodentodigital dysplasia locus: analysis of thirteen candidate genes and identification of novel ESTs and DNA polymorphisms

Oculodentodigital dysplasia (ODDD) is an autosomal dominant condition with congenital anomalies of the craniofacial and limb regions and neurodegeneration. Genetic anticipation for the dysmorphic and neurologic features has been inferred in a few families. Our previous linkage studies have refined the ODDD candidate region to chromosome 6q22→q23. In an attempt to clone the ODDD gene, we created a yeast artificial chromosome contig with 31 redundant clones spanning the region and identified and ordered candidate genes and markers. Fluorescent in situ hybridization mapped two of these YAC clones to chromosome 6q22.2 telomeric to a known 6q21 fragile site, excluding it as a possible cause of the suggested anticipation. We performed mutation analysis on thirteen candidate genes – GRIK2, HDAC2, COL10A1, PTD013, KPNA5, PIST, ROS1, BRD7, PLN, HSF2, PKIB, FABP7, and HEY2. Although no mutations were found, we identified 44 polymorphisms, including 28 single nucleotide polymorphisms. Direct cDNA selection was performed and fifty-five clones were found to contain sequences that were not previously reported as known genes or ESTs. These clones and polymorphisms will assist in the further characterization of this region and identification of disease genes.