Lawrence N. Hjelm

A prevalent mutation for galactosemia among black Americans

OBJECTIVE To define the mutation causing galactosemia in patients of black American origin who have no galactose-1-phosphate uridyltransferase (GALT) activity in erythrocytes but good clinical outcome. METHODS We discovered a mutation caused by a C-->T transition at base-pair 1158 of the GALT gene that results in a serine-to-leucine substitution at codon 135 (S135L). We developed a method with which to screen populations for its prevalence. We compared galactose-1-phosphate uridyltransferase among erythrocytes, leukocytes, and transformed lymphoblasts, as well as total body oxidation of D-(13C)-galactose to 13CO2 among three genotypes for GALT (S135L/S135L, Q188R/Q188R, and Normal/Normal). RESULTS We found a 48% prevalence of the S135L mutation among 17 black American patients with classic galactosemia and a 1% prevalence in a population of 50 black Americans without galactosemia. The S135L mutation was not found in 84 white patients with G/G galactosemia nor in 87 white control subjects without galactosemia. We found normal whole body oxidation of D-(13C)-galactose by the patient homozygous for S135L and various degrees of enzyme impairment among different tissues. CONCLUSIONS The S135L mutation in the GALT gene is a prevalent cause of galactosemia among black patients. Because GALT activity varies in different tissues of patients homozygous for S135L, they may have a better clinical outcome than patients who are homozygous for Q188R when both are treated from infancy.

Mosaic FMR1 Deletion Causes Fragile X Syndrome and Can Lead to Molecular Misdiagnosis : A Case Report and Review of the Literature

The most common cause of fragile X syndrome is expansion of a CGG trinucleotide repeat in the 5′UTR of FMR1. This expansion leads to transcriptional silencing of the gene. However, other mutational mechanisms, such as deletions of FMR1, also cause fragile X syndrome. The result is the same for both the expansion mediated silencing and deletion, absence of the gene product, FMRP. We report here on an 11‐year‐old boy with a cognitive and behavioral profile with features compatible with, but not specific to, fragile X syndrome. A mosaic deletion of 1,013,395 bp was found using high‐density X chromosome microarray analysis followed by sequencing of the deletion breakpoints. We review the literature of FMR1 deletions and present this case in the context of other FMR1 deletions having mental retardation that may or may not have the classic fragile X phenotype.

A molecular approach to galactosemia

Classical galactosemia (G/G) is caused by the lack of galactose-1-phosphate uridyltransferase (GALT) activity. A more common clinical variant, Duarte/Classical (D/G) produces partial enzymatic impairment. Although neonatal death due to G/G galactosemia has been largely eliminated by populationbased screening and intervention, long-term outcome in some is associated with impaired growth, ovarian failure, dyspraxic speech and neurologic deficits. At least 32 variants in the nucleotide sequence of the GALT gene have been identified and 9 have transferred impaired GALT activity to transformed cells in transfection experiments. We here define the prevalence and biochemical phenotype of two mutations. An A to G transition in exon 6 of the GALT gene converts a predicted glutamine at codon 188 to an arginine (Q188R), and introduces a new HpaII cut site into the gene which enables population screening by polymerase chain reaction. An A to G transition in exon 10 in the GALT gene produces a codon change converting an asparagine to aspartic acid at codon 314 (N314D) and adds an AVA II cut site. We screened a large population for the Q188R and N314D sequence changes to investigate the prevalence of Q188R in G/G galactosemia, the effect of homozygosity for Q188R on outcome, and the prevalence and biochemical phenotype of the N314D sequence change. We found that the Q188R mutation has a prevalence of 62% in a predominately Caucasian population of 107 patients with G/G galactosemia. homozygosity for Q188R was associated with a poor clinical outcome in a subgroup of these patients. The N314D mutation is associated with the Duarte biochemical phenotype with extraordinary concordance.

The Duarte allele impairs biostability of galactose‐1‐phosphate uridyltransferase in human lymphoblasts

The Duarte allele (D) is a missense mutation (N314D) that produces a characteristic isoform and partial impairment of galactose‐1‐phosphate uridyltransferase (GALT) in human erythrocytes, fibroblasts, and transformed lymphoblasts. The position of this amino acid is distant, however, from presumptive catalytic site(s) as deduced from a three‐dimensional model of crystallized Escherichia coli galT protein. To evaluate the mechanism(s) involved in the partial impairment of enzymatic activity, we compared the activity, abundance, biological stability, and mRNA of GALT in human lymphoblastoid cell lines cultured from individuals homozygous for wild‐type (WT/WT) and Duarte alleles (N314D/N314D). No other nucleotide differences were present in their GALT genes. The apparent Vmax was reduced in N314D/N314D cells to 31 ± 3.6 compared to WT/WT of 54 ± 6.5 nmole UDP‐galactose formed/g cell protein/hour. Both genotypes had similar apparent KMs for UDP‐glucose of 0.142 ± 0.057 mM and 0.133 ± 0.056 mM. This reduced Vmax was associated with a reduced abundance of the 86kD GALT dimer as determined by Western blots and densitometry. Using RNase protection assays, this reduced GALT protein in the N314D/N314D cell lines was not associated with reduced abundance of GALT mRNA. Using cycloheximide (3‐[2‐(3,5‐Dimethyl‐2‐oxocyclohexyl)‐2‐hydroxyethyl]glutarimide) inhibition of de novo protein synthesis, GALT enzyme activity, and its dimeric protein had a biological T1/2 of ˜24 hours in N314D/N314D cell lines as compared to 50 hours for WT/WT lymphoblasts. Upon exposure to 50°C for 15 minutes, N314D/N314D lymphoblasts retained 45% of GALT activity, whereas controls retained 77% activity. Reduced activity and thermal sensitivity caused by the N314D mutation reverted to control values when a lysine was substituted for a glutamic acid at amino acid 203 in cis (E203K). In summary, N314D/N314D lymphoblasts have reduced GALT enzyme capacity, dimeric protein abundance, biological, and thermal stability. We conclude that the substitution of aspartate for asparagine at amino acid 314 in the human GALT protein reduces the biostability of the active enzyme in human lymphoblasts. Hum Mutat 11:28–38, 1998.

Characterization of an unusual deletion of the galactose-1-phosphate uridyl transferase ( GALT ) gene

Purpose: We previously reported a deletion of the Galactose-1-Phosphate Uridyl Transferase (GALT) gene. This deletion can cause apparent homozygosity for variants located on the opposite allele, potentially resulting in a discrepancy between the biochemical phenotype and the apparent genotype in an individual. The purpose of this study was to determine the deletion breakpoints, allowing the development of a rapid and reliable molecular test for the mutation.Methods: A Polymerase Chain Reaction walking strategy was used to map the 5′ and 3′ breakpoints. The junction fragment was amplified and sequenced to precisely characterize the deletion breakpoints.Results: The deletion has a bipartite structure involving two large segments of the GALT gene, while retaining a short internal segment of the gene. Molecular characterization allowed the development of a deletion specific Polymerase Chain Reaction-based assay. In 25 individuals who had a biochemical carrier galactosemia phenotype, but tested negative for 8 common GALT gene variants, 3 carried this deletion.Conclusion: This deletion occurs at an appreciable frequency and should be considered when there is a discrepancy between the genotype and biochemical phenotype. Many of the individuals carrying the allele were of Ashkenazi Jewish ancestry suggesting that the deletion may be a common cause of galactosemia in that population.

Allelic Dropout Can Cause False-Positive Results for Prader-Willi and Angelman Syndrome Testing

The diagnosis of many genetic disorders relies on a combination of clinical suspicion and confirmatory genetic testing. Our laboratory uses a standard methylation-sensitive PCR (MSP) to target the differentially methylated SNRPN gene to test for Prader-Willi syndrome (PWS) and Angelman syndrome. One patient, a 27-month-old female, who lacked the classical clinical features of PWS, but had a molecular diagnosis of PWS by MSP by another laboratory, had repeat testing in our laboratory. Testing by MSP in our laboratory also identified an apparent loss of the unmethylated paternal allele, consistent with a diagnosis of PWS. Confirmatory testing using Southern blot analysis with a methylation-sensitive restriction enzyme showed a normal pattern of methylation, detecting both the methylated maternal and unmethylated paternal alleles. To investigate these discrepant results, we amplified and sequenced the SNRPN locus in this patient and identified a single nucleotide change within the binding site for the unmethylated DNA-specific primer. These results indicate this nucleotide change led to allelic dropout in the MSP analysis, yielding the false-positive result. Subsequently, MSP analysis using an alternate primer set that was developed by our laboratory detected both methylated and unmethylated alleles. These findings illustrate that allelic dropout due to the presence of rare polymorphisms can cause false-positive results in commonly used MSP assays and lead to molecular misdiagnosis.

Parent-of-Origin Testing for 15q11-q13 Gains by Quantitative DNA Methylation Analysis

The Prader-Willi/Angelman syndrome critical region (PWS/ASCR), located at chromosome 15q11-q13, is associated with several diseases. Absence of paternally expressed genes in this region cause Prader-Willi syndrome (PWS), whereas absence of the maternally expressed UBE3A gene causes Angelman syndrome (AS). In addition, duplications and triplications of this region are also associated with distinct clinical features, indicating that the overexpression of genes within the PWS/ASCR can also lead to distinct phenotypes. Maternally inherited increases in copy number generally lead to a more severe phenotype do than paternally inherited increases. We describe a real-time methylation-sensitive PCR (Q-MSP) assay that quantifies methylation at the promoter of the differentially methylated SNRPN gene located within the PWS/ASCR. Q-MSP can detect both PWS and AS, as well as determine the parent of origin for the allele that carries the PWS/ASCR gains. In addition, Q-MSP requires only a small amount of DNA, is amenable to high-throughput analysis, and can be used in clinical testing as a reflex test to determine the parent of origin after identification of a gain of this region on chromosome 15.

A Simple Method to Confirm and Size Deletion, Duplication, and Insertion Mutations Detected by Sequence Analysis

Characterizing heterozygous insertions or deletions in genes by PCR and Sanger sequencing can be a challenge due to overlapping sequencing traces produced by overlapping templates. This is particularly problematic for clinical diagnostic laboratories, because mutations must be precisely characterized. Although the mutation detection software used by clinical diagnostic laboratories reliably identifies small insertions and deletions, overlapping deletions and insertions on opposite chromosomes, complex rearrangements, and insertions or deletions close to the primer sites may be missed. Here we describe a rapid, simple method to confirm and precisely characterize deletions and insertions using a capillary-based gel electrophoresis system. This technique has been applied to a series of patients with deletion, duplication, or insertion mutations identified by sequencing, as well as to patients with repeat tract polymorphisms, to demonstrate the utility of this method.