Angelos Alexandrou

Implementation of High Resolution Whole Genome Array CGH in the Prenatal Clinical Setting: Advantages, Challenges, and Review of the Literature

Array Comparative Genomic Hybridization analysis is replacing postnatal chromosomal analysis in cases of intellectual disabilities, and it has been postulated that it might also become the first-tier test in prenatal diagnosis. In this study, array CGH was applied in 64 prenatal samples with whole genome oligonucleotide arrays (BlueGnome, Ltd.) on DNA extracted from chorionic villi, amniotic fluid, foetal blood, and skin samples. Results were confirmed with Fluorescence In Situ Hybridization or Real-Time PCR. Fifty-three cases had normal karyotype and abnormal ultrasound findings, and seven samples had balanced rearrangements, five of which also had ultrasound findings. The value of array CGH in the characterization of previously known aberrations in five samples is also presented. Seventeen out of 64 samples carried copy number alterations giving a detection rate of 26.5%. Ten of these represent benign or variables of unknown significance, giving a diagnostic capacity of the method to be 10.9%. If karyotype is performed the additional diagnostic capacity of the method is 5.1% (3/59). This study indicates the ability of array CGH to identify chromosomal abnormalities which cannot be detected during routine prenatal cytogenetic analysis, therefore increasing the overall detection rate. In addition a thorough review of the literature is presented.

FoSTeS, MMBIR and NAHR at the human proximal Xp region and the mechanisms of human Xq isochromosome formation

The recently described DNA replication-based mechanisms of fork stalling and template switching (FoSTeS) and microhomology-mediated break-induced replication (MMBIR) were previously shown to catalyze complex exonic, genic and genomic rearrangements. By analyzing a large number of isochromosomes of the long arm of chromosome X (i(Xq)), using whole-genome tiling path array comparative genomic hybridization (aCGH), ultra-high resolution targeted aCGH and sequencing, we provide evidence that the FoSTeS and MMBIR mechanisms can generate large-scale gross chromosomal rearrangements leading to the deletion and duplication of entire chromosome arms, thus suggesting an important role for DNA replication-based mechanisms in both the development of genomic disorders and cancer. Furthermore, we elucidate the mechanisms of dicentric i(Xq) (idic(Xq)) formation and show that most idic(Xq) chromosomes result from non-allelic homologous recombination between palindromic low copy repeats and highly homologous palindromic LINE elements. We also show that non-recurrent-breakpoint idic(Xq) chromosomes have microhomology-associated breakpoint junctions and are likely catalyzed by microhomology-mediated replication-dependent recombination mechanisms such as FoSTeS and MMBIR. Finally, we stress the role of the proximal Xp region as a chromosomal rearrangement hotspot.

A single gene deletion on 4q28.3: PCDH18--a new candidate gene for intellectual disability?

We report a boy with severe developmental delay, seizures, microcephaly, hypoplastic corpus callosum, internal hydrocephalus and dysmorphic features (narrow forehead, round face, deep-set eyes, blue sclerae, large and prominent ears, nose with anteverted nares, thin upper lip, small and wide-spaced teeth, hyperextensibility of the elbows, wrists, and fingers, fingertip pads, broad hallux, sacral dimple), carrying a 1.53 Mb interstitial deletion at 4q28.3. The deletion was detected by Agilent 105K oligo-array genome hybridization and involves the genomic region between 137,417,338 and 138,947,282 base pairs on chromosome 4 (NCBI build 36). The alteration was inherited from a healthy mother and contains a single gene, PCDH18 which encodes a cadherin-related neuronal receptor thought to play a role in the establishment and function of cell-cell connections in the brain. Thus, haploinsufficiency of this gene may contribute to altered brain development and associated malformations. We found that this deletion is a private inherited copy number variation that is associated with specific clinical findings in our patient and propose the PCDH18 gene as a possible candidate gene for intellectual disability.

Duplication of exons 3–10 of the HSD17B3 gene: A novel type of genetic defect underlying 17β-HSD-3 deficiency

The clinical, biochemical and genetic features of a Cypriot origin male of non-consanguineous parents due to 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD-3) deficiency are presented. The patient, currently a 10 old male, was referred to our clinic because of ambiguous genitalia at birth. Gonads were palpable in the inguinal canal bilaterally and no Müllerian structures identified on pelvic ultrasound. Chromosomal analysis showed an apparently normal male 46,XY karyotype. Diagnosis of 17β-HSD-3 deficiency in the newborn was suspected based on biochemical findings, following human chorionic gonadotrophin (hCG) stimulation test. Sequence analysis and real time PCR along with MLPA identified the patient with a novel 11.96 kb duplication that spans exons 3-10 of the HSD17B3 gene and extends from intron 2 to intron 10 in compound heterozygosity with the known p.R80Q missense mutation leading to 17β-HSD-3. In conclusion, 17β-HSD-3 deficiency was diagnosed in this patient based on endocrinologic evaluation and confirmed with genetic analysis of the HSD17B3 gene. The novel large duplication spanning exons 3-10 of the HSD17B3 gene that we report here in compound heterozygosity with the known p.R80Q leads to 17β-HSD-3 deficiency presenting as 46,XY Disorder of Sex Development. Following diagnosis and appropriate genetic counselling, the patient was raised a boy and successfully underwent surgical correction of crytptorchidism and hypospadias.

Partial MEF2C deletion in a Cypriot patient with severe intellectual disability and a jugular fossa malformation: Review of the literature

Deletions or intragenic mutations involving the MEF2C gene on chromosome 5q14.3 have generally been associated with a relatively uniform phenotype characterized by severe developmental delay, absent speech, stereotypies, absent or limited gait abilities, lack of a typical facial gestalt and scarcity of major malformations. We report on a patient of Cypriot descent with a de novo, approximately 147 kb in size, partial MEF2C deletion removing exons 1 to 3. He had a history of severe intellectual disability with absent speech, poor eye contact, hand stereotypies and a wide‐based gait. A broad‐based, shallow jugular pit with an overlying vascular malformation was also present. Partial MEF2C deletions have only been reported in a very small number of patients and have on occasion been associated with relatively milder phenotypes. We present a patient of Cypriot descent with such a deletion and review previously published literature on partial MEF2C gene deletions postulating a key role of the first few exons in the pathogenesis of the disease.

Alpha‐thalassemia intellectual disability: variable phenotypic expression among males with a recurrent nonsense mutation – c.109C>T (p.R37X)

Alpha‐thalassemia intellectual disability, one of the recognizable X‐linked disability syndromes, is characterized by short stature, microcephaly, distinctive facies, hypotonic appearance, cardiac and genital anomalies, and marked skewing of X‐inactivation in female carriers. With the advent of next generation sequencing, mutations have been identified that result in less severe phenotypes lacking one or more of these phenotypic manifestations. Here we report five unrelated kindreds in which a c.109C>T (p.R37X) mutation segregates with a variable but overall milder phenotype. The distinctive facial appearance of alpha‐thalassemia intellectual disability was present in only one of the 18 affected males evaluated beyond the age of puberty, although suggestive facial appearance was present in several during infancy or early childhood. Although the responsible genetic alteration is a nonsense mutation in exon 2 of ATRX, the phenotype appears to be partially rescued by the production of alternative transcripts and/or other molecular mechanisms.

A prenatally ascertained, maternally inherited 14.8 Mb duplication of chromosomal bands Xq13.2-q21.31 associated with multiple congenital abnormalities in a male fetus.

Duplications of the X chromosome are rare cytogenetic findings, and have been associated with an abnormal phenotype in the male offspring of apparently normal or near normal female carriers. We report on the prenatal diagnosis of a duplication on the long arm of chromosome X from chromosomal band Xq13.2 to q21.31 in a male fetus with increased nuchal translucency in the first trimester and polyhydramnios at 22 weeks of gestation. Amniocentesis was undertaken and cytogenetic analysis revealed additional chromosomal material in the long arm of chromosome X at position Xq13. Analysis with high resolution array CGH revealed the additional material is in fact a duplication of the region Xq13.2-q21.13. The duplication is 14.8 Mb in size and includes fourteen genes: SLC16A2, KIAA2022, ABCB7, ZDHHC15, ATRX, MAGT1, ATP7A, PGK1, TBX22, BRWD3, POU3F4, ZNF711, POF1B and CHM. Analysis of the parents revealed the mother to be a carrier of the same duplication. After elected termination of the pregnancy at 28 weeks a detailed autopsy of the fetus allowed for genotype-phenotype correlations.

Genetic screening of EXT1 and EXT2 in Cypriot families with hereditary multiple osteochondromas.

The purpose of this study was to perform genetic screening of the exostosin 1 (EXT1) and exostosin 2 (EXT2) genes in Cypriot patients with a clinical diagnosis of hereditary multiple osteochondromas (HMO). Initially, mutation analysis of the EXT1 gene was performed by Sanger sequencing. When no point mutation was identified in EXT1, EXT2 analysis was performed. When no sequence variant was identified in either of the candidate genes, array-comparative genomic hybridization (CGH) was implemented to detect any large copy number changes (CNCs). In total, three point mutations and a large deletion were identified in EXT1: the c.2101C>T(p.Arg701*) mutation and the ∼0.16-Mb deletion removing exons 2–11, which were previously reported whereas the c.486_489delCAGA(p.Asp162Glyfs*12) and the c.868G>T(p.Glu290*) mutations which were novel. It is the first report on genetic screening of five HMO patients of Cypriot descent. The observations presented provide additional evidence for the variability in phenotypic expression and the mutational spectrum of this disorder. The term HMO, or hereditary multiple exostoses (HME), MIM: 133700 and MIM: 133701 respectively is used to describe a genetic disorder characterized by the development of multiple, circumscript, occasionally painful and usually symmetric bony protuberances called osteochondromas (benign cartilaginous tumours which most frequently grow outward from the juxtaphyseal region of the long bones or

Mild Phenotype in a Patient with a De Novo 6.3 Mb Distal Deletion at 10q26.2q26.3.

We report on a 29-year-old Greek-Cypriot female with a de novo 6.3 Mb distal 10q26.2q26.3 deletion. She had a very mild neurocognitive phenotype with near normal development and intellect. In addition, she had certain distinctive features and postural orthostatic tachycardia. We review the relevant literature and postulate that certain of her features can be diagnostically relevant. This report illustrates the powerful diagnostic ability of array-CGH in the elucidation of relatively mild phenotypes.

Identification of an AVP-NPII mutation within the AVP moiety in a family with neurohypophyseal diabetes insipidus: review of the literature

Familial neurohypophyseal diabetes insipidus (FNDI) is a disorder characterized by excess excretion of diluted urine (polyuria) and increased uptake of fluids (polydipsia). The disorder is caused by mutations affecting the AVP-NPII gene, resulting in absent or deficient secretion of the antidiuretic hormone arginine vasopressin (AVP) by the neurohypophysis. In this study we examined a three-generation Cypriot kindred suspected to have FNDI. Direct sequencing analysis of AVP-NPII identified a missense mutation (NM_000490.4:c.61T>C; p.Tyr21His; rs121964893) within the AVP moiety on exon 1 of the gene in all affected family members. So far, only three studies have reported mutations within the AVP moiety of AVP-NPII as being associated with FNDI, with the vast majority of identified FNDI mutations being located within the signalling peptide or the neurophysis II (NPII) moiety of the gene. The mutation within the AVP moiety identified here had been reported previously in a Turkish kindred with FNDI. Consequently, the findings of this study confirm the causal role of mutations within the AVP moiety in FNDI. Herein we review reported mutations within the AVP moiety of AVP-NPII and their contribution to FNDI.