Anthi Drousiotou

Evidence for balancing selection from nucleotide sequence analyses of human G6PD.

Glucose-6-phosphate dehydrogenase (G6PD) mutations that result in reduced enzyme activity have been implicated in malarial resistance and constitute one of the best examples of selection in the human genome. In the present study, we characterize the nucleotide diversity across a 5.2-kb region of G6PD in a sample of 160 Africans and 56 non-Africans, to determine how selection has shaped patterns of DNA variation at this gene. Our global sample of enzymatically normal B alleles and A, A-, and Med alleles with reduced enzyme activities reveals many previously uncharacterized silent-site polymorphisms. In comparison with the absence of amino acid divergence between human and chimpanzee G6PD sequences, we find that the number of G6PD amino acid polymorphisms in human populations is significantly high. Unlike many other G6PD-activity alleles with reduced activity, we find that the age of the A variant, which is common in Africa, may not be consistent with the recent emergence of severe malaria and therefore may have originally had a historically different adaptive function. Overall, our observations strongly support previous genotype-phenotype association studies that proposed that balancing selection maintains G6PD deficiencies within human populations. The present study demonstrates that nucleotide sequence analyses can reveal signatures of both historical and recent selection in the genome and may elucidate the impact that infectious disease has had during human evolution.

Magnetic Resonance Imaging Findings and Novel Mutations in GM1 Gangliosidosis

Two unrelated children and their siblings of Arab origin were diagnosed as having GM1 gangliosidosis on the basis of clinical features and markedly low levels of β-galactosidase. The T2-weighted magnetic resonance images of the brain revealed certain characteristic features, including delayed myelination and abnormal appearance of the subcortical white matter, internal capsule, and basal ganglia. Their mutation analysis showed two novel mutations, which have not been described in an Arabic population. (J Child Neurol 2005;20:57—60).

Identifying Non–Duchenne Muscular Dystrophy–Positive and False Negative Results in Prior Duchenne Muscular Dystrophy Newborn Screening Programs: A Review

IMPORTANCE Duchenne muscular dystrophy (DMD) is a candidate for the recommended universal screening panel based on evidence that early corticosteroid treatment improves outcomes and on new genetic therapies that require early diagnosis for effectiveness. Elevated creatine kinase levels in the neonatal period are the initial screening marker in DMD newborn screening programs but is found in inherited muscle disorders other than DMD. Data are needed to inform protocols for future screening and follow-up testing and care in these patients. OBJECTIVES To review non-DMD muscle disorders identified by prior DMD screening programs and to investigate whether these programs failed to identify patients later diagnosed as having DMD (false-negative findings). EVIDENCE REVIEW Since 1975, 10 DMD newborn screening programs have provided opportunities to study screening protocols, outcomes, and parental responses. These programs used elevated creatine kinase levels in dried blood spots for the initial screening, with the diagnosis of DMD based on findings of clinical follow-up, muscle biopsy, or direct mutational testing of the DMD gene. Literature regarding these prior programs was reviewed in PubMed, and the programs were discussed directly with the directors when possible to identify diagnoses of non-DMD disorders and false negative results from 1975 to July 12, 2015. Data were collected from screening programs, which were active between 1975 and December 2011. Data were analyzed from March 26, 2015, to August 24, 2015. FINDINGS The 10 screening programs screened more than 1.8 million newborns between 1975 and 2011, and 344 were diagnosed with DMD. Of those screened, the majority were boys. Across all programs, 80 patients had positive results for non-DMD disorders, including Becker muscular dystrophy and forms of limb-girdle and congenital muscular dystrophies, and 21 patients had false-negative findings for DMD. CONCLUSIONS AND RELEVANCE Screening for DMD will result in identification of other muscle diseases. Future screening protocols should include infants of both sexes and include follow-up testing algorithms to evaluate patients who do not have DMD gene mutations but may have another muscle disorder associated with elevated neonatal creatine kinase levels. These programs will need to be aware that false-negative results are a possibility.

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 β-hexosaminidase β 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 patients 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.

Hb Agrinio [α29(B10)Leu→Pro (α2)] in Combination with – –MED I Results in a Severe Form of Hb H Disease

We report two cases of compound heterozygote patients for the – –MED I and Hb Agrinio [α29(B10)Leu→Pro (α2)] anomalies in two unrelated Greek Cypriot families. The first patient had a serious form of Hb H disease and died at the age of 21 due to complications arising during an operation. The second patient showed a severe hematological picture and has been regularly transfused since an early age. This patient exhibits bone abnormalities as well as hepatosplenomegaly. The severity of these two incidences emphasizes the need for the inclusion of a screening test for the – –MED I/αAgrinioα genotype among those already offered during prenatal diagnosis. Two homozygotes, as well as a number of simple, compound, and double heterozygotes for Hb Agrinio have been identified in Cyprus and their hematological indices are presented.

Beta thalassaemia intermedia due to co-inheritance of three unique alpha globin cluster duplications characterised by next generation sequencing analysis

Co-inheritance of a thalassaemia reduces chain imbalance and disease severity in b thalassaemia homozygotes, while increasing a globin output in heterozygotes increases chain imbalance, converting a typically asymptomatic carrier state to that of thalassaemia intermedia. The outcome depends on the number of a globin genes inherited as one or two copies of triplicated (/aaa), or quadruplicated (/aaaa) a globin complexes, and the type of b thalassaemia mutation (b or b) (Thein, 2008). Another mechanism of increasing a globin output is through segmental duplication of the whole a globin complex (Harteveld et al, 2008) but breakpoints of the reported duplications have not been fully characterised due to technological limitations. Here, we applied a previously described next generation sequencing (NGS) methodology (Shooter et al, 2015a,b) to characterise three a globin cluster duplications, permitting to-the-base resolution in two of the three cases. Patient samples were referred for work-up of unusually severe phenotype in b thalassaemia carriers. In Family 1 (Fig 1A), the proband was a 54-year-old Chinese male with hypochromic microcytic anaemia since infancy. His partner (Anglo-Saxon English) and two older sons had normal haematological profiles; the youngest son (aged 14 years) had a haematological profile similar to that of the father. Both father and son had

Mouse Stbd1 is N-myristoylated and affects ER-mitochondria association and mitochondrial morphology

ABSTRACT Starch binding domain-containing protein 1 (Stbd1) is a carbohydrate-binding protein that has been proposed to be a selective autophagy receptor for glycogen. Here, we show that mouse Stbd1 is a transmembrane endoplasmic reticulum (ER)-resident protein with the capacity to induce the formation of organized ER structures in HeLa cells. In addition to bulk ER, Stbd1 was found to localize to mitochondria-associated membranes (MAMs), which represent regions of close apposition between the ER and mitochondria. We demonstrate that N-myristoylation and binding of Stbd1 to glycogen act as major determinants of its subcellular targeting. Moreover, overexpression of non-myristoylated Stbd1 enhanced the association between ER and mitochondria, and further induced prominent mitochondrial fragmentation and clustering. Conversely, shRNA-mediated Stbd1 silencing resulted in an increase in the spacing between ER and mitochondria, and an altered morphology of the mitochondrial network, suggesting elevated fusion and interconnectivity of mitochondria. Our data unravel the molecular mechanism underlying Stbd1 subcellular targeting, support and expand its proposed function as a selective autophagy receptor for glycogen and uncover a new role for the protein in the physical association between ER and mitochondria. Highlighted Article: The glycogen-binding protein Stbd1 is N-myristoylated and targeted to ER–mitochondria contact sites. Stbd1 loss- or gain-of-function affects ER–mitochondria association and mitochondrial morphology.

A Novel Large Deletion Encompassing the Whole of the Galactose-1-Phosphate Uridyltransferase ( GALT ) Gene and Extending into the Adjacent Interleukin 11 Receptor Alpha ( IL11RA ) Gene Causes Classic Galactosemia Associated with Additional Phenotypic Abnormalities

Objective The characterization of a novel large deletion in the galactose-1-phosphate uridyltransferase (GALT) gene accounting for the majority of disease alleles in Cypriot patients with classic galactosemia. Methods DNA sequencing was used to identify the mutations followed by multiplex ligation-dependent probe amplification (MLPA) analysis in the cases suspected of harboring a deletion. In order to map the breakpoints of the novel deletion, a PCR walking approach was employed. A simple PCR assay was validated for diagnostic testing for the new deletion. Haplotype analysis was performed using microsatellite markers in the chromosomal region 9p. RT-PCR was used to study RNA expression in lymphoblastoid cell lines. Results The new deletion spans a region of 8489 bp and eliminates all GALT exons as well as the non-translated sequences of the adjacent interleukin 11 receptor alpha (IL11RA) gene. In addition, the deletion is flanked by a 6 bp block of homologous sequence on either side suggesting that a single deletion event has occurred, probably mediated by a recombination mechanism. Microsatellite marker analysis revealed the existence of a common haplotype. The RNA expression studies showed a lack of IL11RA transcripts in patients homozygous for the deletion. Conclusions We have identified and characterized a novel contiguous deletion which affects both the GALT enzyme and the IL11RA protein resulting in classic galactosemia with additional phenotypic abnormalities such as craniosynostosis, a feature that has been associated with defects in the IL11RA gene.

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 β‐subunit of β‐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. Hum Mutat 13:38–43, 1999.

Minimally symptomatic mcardle disease, expanding the genotype–phenotype spectrum

Introduction: We report the clinical, biochemical, and molecular findings in a Cypriot family with minimally symptomatic McArdle disease. Methods: Myophosphorylase in muscle was assessed by histochemistry, quantitative spectrophotometry, and western blot analysis. Mutation identification was performed by PCR amplification of all PYGM exons, followed by bidirectional sequencing. Screening for the new mutation was performed by restriction enzyme analysis. Results: We found that a novel c.1151C>T transition in exon 10 of the myophosphorylase gene (PYGM) is associated with minimally symptomatic McArdle disease. Homozygous carriers displayed an ischemic exercise response characterized by a blunted increase in post‐exercise blood lactate levels in conjunction with an exaggerated increase in ammonia. Myophosphorylase activity in muscle was 3.75% of normal, whereas the size and abundance of the enzyme were unaffected. Conclusions: These findings expand the genotype–phenotype spectrum of McArdle disease and suggest that enzymatic activity as low as 4% may be sufficient to ameliorate the phenotype. Muscle Nerve 52: 891–895, 2015