Barry Eng

Anomalous migration of PCR products using nondenaturing polyacrylamide gel electrophoresis: the amelogenin sex-typing system.

Sex-typing of biological samples can be accomplished using the polymerase chain reaction (PCR) to amplify DNA sequences that are specific for the Y-chromosome. One such system is based on PCR amplification of the X-chromosome amelogenin gene and the amelogenin-like sequences located near the centromere of the Y-chromosome. The X and Y PCR products can be distinguished from each other on the basis of a 177 basepair (bp) insertion in the X relative to the Y. In this report, we demonstrate that the amelogenin PCR products migrate anomalously using non-denaturing polyacrylamide gel electrophoresis (ND-PAGE) as opposed to agarose gel electrophoresis or denaturing PAGE. These results may be relevant to the choice of electrophoretic system used to analyze highly polymorphic loci for individual identification.

Filipino β-thalassemia due to a large deletion: identification of the deletion end points and polymerase chain reaction (PCR)-based diagnosis

The gene frequency of β-thalassemia among Filipinos is estimated to be 0.02, although little is known about the mutations involved. Recently, an extensive β-thalassemia deletion was reported in several unrelated individuals of Filipino descent. The deletion begins approximately 4 kb upstream of the β-globin gene, and extends 3′ beyond the β-globin gene. In this report, we identify the 5′ and 3′ deletion endpoints and present a polymerase chain reaction (PCR) strategy for rapid DNA diagnosis of the Filipino β-thalassemia deletion.

A rapid and simple electrophoretic method for the detection of mutations involving small insertion or deletion: application to β-thalassemia

SummaryThe 1.8-kb β-globin gene fragments of DNAs from individuals heterozygous for nine different β-thalassemia mutations involving 1, 2, 3, 4, or 25 basepair (bp) insertions or deletions were amplified by the polymerase chain reaction (PCR). The PCR products were subjected to electrophoresis on aqueous 8% polyacrylamide gel. In each heterozygote with either a 2 to 25 bp deletion, but not with a 1 bp insertion, two slower migrating bands representing heteroduplexes in addition to the 1.8-kb homoduplex band were seen. The electrophoretic positions of these slower migrating bands were characteristic of each mutation studied. By co-amplification with known normal DNA, it was also possible to distinguish DNAs from normal individuals and from individuals who are homozygous for the small insertion/deletion mutations. These studies demonstrate that the heteroduplex formation generated in PCR can be applied as a simple method in the diagnosis of insertion/deletion mutations involving 2 to 25 bp in β-thalassemias as well as in other genetic disorders.

PCR‐based diagnosis of the Filipino (−−FIL) and Thai (−−THAI) α‐thalassemia‐1 deletions

In southeast Asia, the carrier frequency of two‐gene α‐thalassemia deletions is quite high, ranging from 4% to 14% depending on the population. The most common α‐thalassemia‐1 deletion is the so‐called southeast Asian deletion (−−SEA). In addition, a significant proportion of cases involve two other deletions, the Filipino (−−FIL) and Thai (−−THAI) deletions. In this report, we identify the deletion breakpoints for the (−−FIL) and (−−THAI) deletions, and describe PCR‐based protocols for rapid and reliable DNA diagnosis of these deletions. Am. J. Hematol. 63:54–56, 2000.

Late-onset metachromatic leukodystrophy clinically presenting as isolated peripheral neuropathy: compound heterozygosity for the IVS2+1G-->A mutation and a newly identified missense mutation (Thr408Ile) in a Spanish family.

We report the case of a 50‐year‐old woman and her 32‐year‐old daughter, both of whom are affected with adult‐onset metachromatic leukodystrophy (MLD) clinically presenting as peripheral neuropathy. Arylsulfatase A (ARSA) activities were markedly reduced, and electrophysiology showed a severe demyelinating neuropathy with features of chronic acquired demyelinating polyneuropathy. Molecular genetic studies of the family revealed that the proband and her affected daughter are compound heterozygotes for the common IVS2+1G→A mutation and a newly identified missense mutation, Thr408Ile. This case indicates that adult metachromatic leukodystrophy should be considered in adult patients with demyelinating peripheral neuropathy of unknown etiology.

De novo mutation of the β‐globin gene initiation codon (ATG→AAG) in a Northern European

We present a case of β‐thalassemia intermedia involving a 13‐year‐old boy of Northern European descent. His mother, father and older sister have normal hematologic indices. Molecular studies demonstrate that the proband carries a novel mutation of the β‐globin gene initiation codon (ATG→AAG) which should give rise to β°‐thalassemia trait. The possibility of non‐paternity was excluded, indicating that the novel mutation was the result of a de novo event. A review of the literature indicates that mutations involving the β‐globin gene initiation codon can give rise to a more severe phenotype than is generally associated with most other β+ or β° mutations. Am. J. Hematol. 56:179–182, 1997.

Identification of an extensive ζ-α globin gene deletion in a Chinese individual

We describe a novel α‐thalassaemia‐1 deletion that removes the entire ζ‐α globin gene cluster. A Chinese couple were referred for counselling after two consecutive pregnancies ended with fetal hydrops. Gene mapping was used to demonstrate that the mother is heterozygous for the South‐east Asia α‐thalassaemia‐1 deletion (ζζα/ζζ—SEA), while the father carries an α‐thalassaemia‐1 deletion of more than 100 kilobases (ζζα/—). This newly discovered deletion extends for unknown distances 3’ and 5’ of the ζ‐α globin gene cluster and has been designated (—HW).

Spectrum of beta-thalassemia mutations in Egypt.

Carrier screening and prenatal diagnosis programs have successfully reduced the incidence of β-thalassemia (thal) in countries where the carrier rates are high (1-3). The incidence of β-thal is particularly high in Egypt (4) due to the combined effects of high carrier rates (5) and consanguineous marriages (6). As a prerequisite to establishing effective programs for prenatal diagnosis, it is important to identify the spectrum of mutations within the population. To this end, we have surveyed the β-thal mutations for a cohort of 55 transfusion-dependent β-thal patients from the Alexandria region of Egypt and the adjacent rural governorates. Affected siblings were not included in the study. The patients were either Moslem (46/55, 84%) or Christian (9/55, 16%). Almost three-quarters (40/55, 73%) of the patients had consanguineous parents.

Attitudes of high school students toward carrier screening and prenatal diagnosis of cystic fibrosis

The identification of the common mutations of the cystic fibrosis (CF) transmembrane conductance regulator gene has made it feasible to consider population-based CF carrier screening. However, the demand for such programs will depend largely on the attitudes and perceptions of the general public toward genetic testing. As part of a high school science project, we initiated studies to determine the attitudes of high school students toward carrier screening for CF and other genetic disorders. From a class of 120 biology students, 101 consented to participate in the study. Most of the students were of European ancestry and there were no known family histories of CF. Buccal swabs were obtained for PCR amplification and heteroduplex detection of the most common CF mutation (ΔF508). The students and their corresponding buccal swabs were assigned random numbers and the results were kept confidential unless the student requested his/her test result. A questionnaire was used to assess the students attitudes toward carrier screening and prenatal diagnosis. This questionnaire was given both before testing and after the class was informed that three individuals were carriers of the ΔF508 mutation. Overall, the data indicate that high school students are receptive to the concepts of carrier screening and prenatal diagnosis. However, their attitudes changed considerably when informed of the possibility that they might actually be a ΔF508 carrier, with marked increases in the level of indecision. At the conclusion of the study, very few students (5/101) requested information about their ΔF508 carrier status.

Atypical clinical course in juvenile metachromatic leukodystrophy involving novel arylsulfatase A gene mutations

A male and female with juvenile metachromatic leukodystrophy (MLD) with unusual manifestations are presented, each involving a novel arylsulfatase A gene mutation. One patient demonstrated acute intermittent encephalopathic episodes for 1 year after having received the diagnosis of MLD at the age of 6 years. The other patient presented at the age of 5 years with acute hemiparesis, which was diagnosed as acute disseminated encephalomyelitis and resolved in 3 weeks. After 2 years of remission he started to show progressive neurological deterioration. The episodic manifestations in both patients were associated with acute, resolving cerebral lesions on magnetic resonance imaging accompanying or preceding the classical demyelinating lesions of MLD. The diagnosis of MLD was based on arylsulfatase A enzyme activity levels and genetic analysis, and after the exclusion of neurological conditions such as encephalitis, vasculopathy, or mitochondrial disorders. The pathogenesis of this previously undescribed finding in MLD is unknown but might be related to a susceptibility of myelin to acute damage.