Ethylmalonic encephalopathy: application of improved biochemical and molecular diagnostic approaches.

Ethylmalonic encephalopathy (EE, OMIM # 602473) is an autosomal recessive metabolic disorder of infancy affecting the brain, the gastrointestinal tract and peripheral vessels. It is caused by a defect in the ETHE1 gene product, which was recently shown to be part of a metabolic pathway devoted to sulphide detoxification. We report the application of improved biochemical and molecular approaches to the diagnosis of three cases of EE from two unrelated Cypriot families. The children presented all the typical biochemical hallmarks of the disease including elevated lactate and butyrylcarnitine in blood and elevated urinary excretion of ethylmalonic acid, 2-methylsuccinate, isobutyrylglycine and isovalerylglycine. We also detected an elevated level of thiosulphate in urine, which we propose as an additional biochemical marker of the disease. The proband of the first family was shown to be a compound heterozygote for a missense mutation in exon 5, L185R, and a deletion of exon 4. The deletion was identified using quantitative real-time polymerase chain reaction (qRT-PCR). Using the same technique, the proband of the second family was found to be homozygous for the exon 4 deletion. A prenatal diagnosis was performed for the second family using qRT-PCR, thus establishing the usefulness of RT-PCR in prenatal diagnosis.

Haplotype diversity and linkage disequilibrium at human G6PD: recent origin of alleles that confer malarial resistance.

The frequencies of low-activity alleles of glucose-6-phosphate dehydrogenase in humans are highly correlated with the prevalence of malaria. These "deficiency" alleles are thought to provide reduced risk from infection by the Plasmodium parasite and are maintained at high frequency despite the hemopathologies that they cause. Haplotype analysis of "A-" and "Med" mutations at this locus indicates that they have evolved independently and have increased in frequency at a rate that is too rapid to be explained by random genetic drift. Statistical modeling indicates that the A- allele arose within the past 3840 to 11,760 years and the Med allele arose within the past 1600 to 6640 years. These results support the hypothesis that malaria has had a major impact on humans only since the introduction of agriculture within the past 10,000 years and provide a striking example of the signature of selection on the human genome.

Pilot Experience with an External Quality Assurance Scheme for Acylcarnitines in Plasma/Serum.

The analysis of acylcarnitines (AC) in plasma/serum is established as a useful test for the biochemical diagnosis and the monitoring of treatment of organic acidurias and fatty acid oxidation defects. External quality assurance (EQA) for qualitative and quantitative AC is offered by ERNDIM and CDC in dried blood spots but not in plasma/serum samples. A pilot interlaboratory comparison between 14 European laboratories was performed over 3 years using serum/plasma samples from patients with an established diagnosis of an organic aciduria or fatty acid oxidation defect. Twenty-three different samples with a short clinical description were circulated. Participants were asked to specify the method used to analyze diagnostic AC, to give quantitative data for diagnostic AC with the corresponding reference values, possible diagnosis, and advice for further investigations.Although the reference and pathological concentrations of AC varied among laboratories, elevated marker AC for propionic acidemia, isovaleric acidemia, medium-chain acyl-CoA dehydrogenase, very long-chain acyl-CoA dehydrogenase, and multiple acyl-CoA dehydrogenase deficiencies were correctly identified by all participants allowing the diagnosis of these diseases. Conversely, the increased concentrations of dicarboxylic AC were not always identified, and therefore the correct diagnosis was not reach by some participants, as exemplified in cases of malonic aciduria and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency. Misinterpretation occurred in those laboratories that used multiple-reaction monitoring acquisition mode, did not derivatize, or did not separate isomers. However, some of these laboratories suggested further analyses to clarify the diagnosis.This pilot experience highlights the importance of an EQA scheme for AC in plasma.

Four novel mutations in patients from the Middle East with the infantile form of GM1-gangliosidosis.

GM1-gangliosidosis is a lysosomal storage disorder caused by a deficiency of beta-galactosidase. It is mainly characterized by progressive neurodegeneration and in its most severe infantile form it leads to death before the age of four. We have performed molecular analysis of five patients with the infantile form of GM1-gangliosidosis originating from the Middle East (two from Saudi Arabia and three from the United Arab Emirates). We have identified four novel mutations and one previously reported mutation in the GLB1 gene. The first novel mutation found in the homoallelic state in a patient from Saudi Arabia, is a c.171C>G transversion in exon 2 which creates a premature stop codon. Northern blot analysis in fibroblasts from the patient showed no mRNA and expression studies in COS-1 cells showed complete absence of the 85kDa precursor protein and no catalytic activity. The second novel mutation is a splicing error in intron 2, c.245+1G>A. This mutation was found in the heteroallelic state in a patient from Saudi Arabia, the second mutation being the previously described c.145C>T mutation. The third novel mutation is a missense mutation in exon 4, c.451G>T, found in the homoallelic state in a patient from the United Arab Emirates. Expression studies of this mutation in COS-1 cells showed complete absence of the 85kDa precursor protein and no catalytic activity. The fourth novel mutation is a splicing mutation in intron 8, c.914+4A>G, found in the homoallelic state in two siblings from the United Arab Emirates. This study has revealed genetic heterogeneity of the beta-galactosidase deficiency in the Arabic population [corrected]

Dystrophinopathy presenting as congenital muscular dystrophy.

We report a 3 1/2-year-old boy with congenital hypotonia, calf pseudohypertrophy, markedly delayed motor milestones and joint contractures. He was initially diagnosed to have congenital muscular dystrophy on the basis of the age of onset, a myopathic EMG, an elevated creatine kinase and a dystrophic muscle biopsy. Subsequently, dystrophin immunocytochemistry and immunoblot analysis showed complete absence of dystrophin. We suggest that male cases of CMD should undergo dystrophin analysis, if there is calf hypertrophy and markedly elevated CK (> 2000 U/l).

Neonatal screening for Duchenne muscular dystrophy: a novel semiquantitative application of the bioluminescence test for creatine kinase in a pilot national program in Cyprus.

The objectives of this study were to evaluate a novel semiquantitative application of the bioluminescence test for screening newborns for Duchenne muscular dystrophy (DMD) and to use this technique in a pilot national program. The study was performed on the island of Cyprus, which provides ideal conditions for maximizing the prevention rate due to the small size of the country, the well-defined population, and the high degree of awareness of the public concerning genetic diseases. Guthrie spots were obtained through the national screening center for phenylketonuria and congenital hypothyroidism. The bioluminescence method for measuring creatine kinase (CK) in dried blood spots was adapted for use in a semiquantitative way. During the first 6 years of the program (1992-1997), we screened 30,014 samples and found 43 with initially high CK values. We were able to obtain repeat specimens in 35 cases. Of the repeat samples, 30 were found to have normal activity, giving a false-positive rate of 0.10%. Five boys had persistent CK elevations and were confirmed to be DMD or Becker (BMD) cases by DNA analysis and/or dystrophin analysis. The semiquantitative application of the bioluminescence assay of CK that we have introduced has proved to be a fast and reliable method for screening large numbers of samples for DMD. It has a low rate of false positives, which compares favorably with that of other DMD screening programs. Although it is early to evaluate its impact fully, the program seems to be bringing about the anticipated benefits to affected families.

A comparative morphological study in 33 cases of respiratory chain encephalomyopathies.

Mitochondrial encephalomyopathies (ME) are clinically and genetically heterogeneous syndromes ranging from pure myopathies to complex multisystem disorders. This phenotypic and genotypic variability, coupled with the lack of a laboratory gold standard marker for the diseases, makes diagnosis a challenging process. Mitochondrial DNA analysis and biochemical assay of muscle homogenates are quite specific diagnostically but have low sensitivity in unselected cases suspected of ME. We decided to evaluate four routine morphological methods in 33 cases of definite or probable ME in an effort to assess the reliability of each of these techniques in diagnosing ME.

Mutation analysis of a Sandhoff disease patient in the Maronite community in Cyprus.

Sandhoff disease occurs in the Christian Maronite community in Cyprus, a community that established over a thousand years ago. Nowadays, this community comprises less than 1% of the whole population, and has been culturally and socially isolated. Cultured fibroblasts from a patient from this inbred group showed a beta-hexosaminidase beta subunit mRNA of apparently the normal size but of reduced quantity. A mutational analysis of cDNA obtained by polymerase chain reaction amplification of mRNA showed a deletion of A at nt 76 (counted from A of the initiation codon, ATG). The deletion results in a frame shift and a premature termination within 20 amino acids from the N-terminus of the normal mature enzyme protein. The patient was homozygous for the deletion. The 5'-end of the gene showed many discrepancies from the previously published sequence. We consider that these differences are probably polymorphisms of little functional significance, because the patient's fibroblasts generate decreased but stable mRNA and because some of these base changes were also found in the genes from control fibroblasts. An extensive evaluation of the prevalence of this mutant allele in this community is being initiated.

Novel splice site mutation at IVS8 nt 5 of HEXB responsible for a Greek-Cypriot case of Sandhoff disease.

Sandhoff disease is caused by abnormalities in HEXB gene encoding the beta-subunit of beta-hexosaminidase. In this study, we analyzed the HEXB gene of a Sandhoff carrier in the Greek-Cypriot community. A G to C transversion was identified in one allele of her HEXB gene at position 5 of the 5'-splice site of intron 8 (IVS8 nt5). One of 13 cDNA clones derived from her lymphocyte HEXB mRNA lacked the last four nucleotides "GTTG" of exon 8, which created a premature termination codon at 11 codons downstream. In vivo transcription of the mutant HEXB gene fragment in CHO cells resulted in deletion of the "GTTG." The mutation has not been found in 40 DNA samples from anonymous donors, indicating that this is not a polymorphism in the Cypriot population. These results clearly indicate that the splice site mutation at IVS8 nt5 is responsible for this case of Sandhoff disease.

Alpha-thalassaemia prenatal diagnosis by two PCR-based methods.

In Cyprus all couples carrying alpha0-thalassaemia mutations are detected in the course of the thalassaemia carrier screening program and prenatal diagnosis is offered to all of them. Prenatal diagnosis for alpha-thalassaemia is routinely done by two independent molecular methods. With the first method, the mutations of the parents are directly determined by gap-PCR and then the chorionic villus sample (CVS) is examined for the presence of these mutations. With the other method, a (CA)n repeat polymorphic site located between the psialpha1- and alpha2-globin genes is used for determining the presence or absence of the normal and mutant alleles. In the period from 1995 to 1999, molecular analysis of 46 couples in which haematological data were consistent with deletion of two alpha-globin genes in both partners indicated that only 13 of them were actually at risk for haemoglobin (Hb) Bart's hydrops fetalis and prenatal diagnosis was provided in 16 pregnancies. The molecular diagnosis was possible in all cases with the use of both gap-PCR and (CA)n repeat polymorphisms analysis. No misdiagnosed cases for alpha-thalassaemia have been reported to date.

Sandhoff disease in Cyprus: population screening by biochemical and DNA analysis indicates a high frequency of carriers in the Maronite community.

In the last 15 years, four patients with the infantile form of Sandhoff disease were diagnosed in four different families in Cyprus (population 703,000, birth rate 1.7%). Three of these cases came from the Christian Maronite community (less than 1% of the population) and one from the Greek community (84% of the population). This relatively large number of patients prompted us to initiate an epidemiological study in order to establish the frequency of the mutant allele in Cyprus. Carrier detection was initially based on the measurement of beta-hexosaminidase A and B in both leucocytes and serum. Using the enzyme test, 35 carriers were identified among 244 random Maronite samples and 15 among 28 Maronites with a family history of Sandhoff disease, but only one carrier was found out of 115 random samples from the Greek community. In parallel to the biochemical screening, DNA studies were undertaken in one of the three Maronite patients and in a Greek carrier related to the Greek patient. These studies resulted in the identification of two novel mutations, a deletion of A at nt76 and a G to C transversion at position 5 of the 5'-splice site of intron 8, which have been published. We subsequently screened the carriers detected in the biochemical study for these two mutations using PCR-based tests. Of 50 Maronite carriers examined, 42 were found to have the nt76 deletion. Eight Maronite samples, designated carriers from the biochemical results, were negative for both mutations. It is possible that these individuals were incorrectly classified as carriers since their enzyme values are equivocal, although the presence of another mutation has not been excluded. Two Greek Cypriot carriers and two obligate Lebanese carriers were negative for both mutations. We conclude that there is a high frequency of Sandhoff disease carriers in the Maronite community of Cyprus, approximately 1 in 7, and that a single mutation predominates in this population.