[Prenatal diagnosis of trisomy 13 and trisomy 18: the experience of Assaf-Harofe Medical Center].

INTRODUCTION: Trisomy 18 and 13 are the most common autosomal trisomies, after trisomy 21, and their frequency is rising due to the increased maternal age of pregnant women. The fetuses suffer from multi-organ damage that may lead to many gestational complications as well as short life expectancy. OBJECTIVE: To assess the indications for prenatal karyotyping of trisomy 13 (T-13, Patau syndrome) and trisomy 18 (T-18, Edwards syndrome) during pregnancy in our medical center. METHODS: This retrospective cohort study involved all singleton pregnancies locally diagnosed or referred to our Institute because of T-13 and T-18, during the years 1998-2011. RESULTS: There were 1879 cases of termination of pregnancies (TOPs) because of fetal indications, of them 53 cases of T-18 and 10 cases of T-13. The main indications for prenatal karyotyping in our study group were abnormal sonographic findings during anomaLy scans. In addition, 7 newborns with T-18 and 3 infants with T-13 were born in our hospital during the same period of time. We examined all cases that led to the Live birth of newborns with chromosomal anomalies, stemming from the Lack of extraction of the tests mentioned above and/or ignoring findings that raise suspicion that requires performing prenatal karyotyping during pregnancy. DISCUSSION: Our findings corresponded with other studies and showed that prenatal diagnosis of T-13/T-18 due to abnormal sonographic finding is rising. CONCLUSIONS: Our study shows that it was possible to identify the vast majority of T-13/T-18 among the pregnant women who had an increased risk based on a combination of the routine screening tests applied in Israel.

Screening and cell-based assessment of mutations in the Aristaless-related homeobox (ARX) gene.

ARX mutations cause a diverse spectrum of human disorders, ranging from severe brain and genital malformations to non-syndromic intellectual disability (ID). ARX is a transcription factor with multiple domains that include four polyalanine (pA) tracts, the first two of which are frequently expanded by mutations. We progressively screened DNA samples from 613 individuals with ID initially for the most frequent ARX mutations (c.304ins(GCG)(7)'expansion' of pA1 and c.429_452dup 'dup24bp' of pA2). Five hundred samples without pA1 or pA2 mutations had the entire ARX ORF screened by single stranded polymorphism conformation (SSCP) and/or denaturing high pressure liquid chromatography (dHPLC) analysis. Overall, eight families with six mutations in ARX were identified (1.31%): five duplication mutations in pA2 (0.82%) with three new clinical reports of families with the dup24bp and two duplications larger than the dup24bp mutation discovered (dup27bp, dup33bp); and three point mutations (0.6%), including one novel mutation in the homeodomain (c.1074G>T). Four ultraconserved regions distal to ARX (uc466-469) were also screened in a subset of 94 patients, with three unique nucleotide changes identified in two (uc466, uc467). The subcellular localization of full length ARX proteins was assessed for 11 variants. Protein mislocalization increased as a function of pA2 tract length and phenotypic severity, as has been previously suggested for pA1. Similarly, protein mislocalization of the homeodomain mutations also correlated with clinical severity, suggesting an emerging genotype vs cellular phenotype correlation.

Sporadic aneuploidy in PHA-stimulated lymphocytes of trisomies 21, 18, and 13.

Following the observation detected in a previous study that X chromosome monosomy in Turner's syndrome genotypes was associated with a sporadic loss and/or gain of other chromosomes, we studied here whether this instability is a consistent finding in constitutional autosomal trisomies. We used PHA-stimulated lymphocytes derived from 14 patients (10 patients with trisomy 21, 2 with trisomy 18, and 2 with trisomy 13). Fourteen healthy controls were compared. Fluorescence in situ hybridization, applied at interphase cells, was used to evaluate the level of aneuploidy for 3 randomly selected chromosomes (autosomes 8, 15, and 16) in each sample. For each tested chromosome, our results showed a significantly higher level of aneuploid cells in the samples from the patients than in those from controls, with no difference between the patient groups. The mean level of aneuploid cells (percentage) for all 3 tested autosomes was almost twice as high in the patient samples as in the control samples. The aneuploidy level was mainly due to monosomy, which was significantly higher in the samples from the patients than in those from controls for each one of the tested chromosomes, with no difference between the patient groups. The mean level of monosomic cells (percentage) for all 3 tested chromosomes was almost twice as high in the patient samples as in the control samples. Our study shows that various constitutional autosomal trisomies are associated with an increased frequency of non-chromosome specific aneuploidy and is a continuation of the previous study documenting sporadic aneuploidy in Turner's sample cells. It is possible that primary aneuploid cells destabilize their own genome resulting in variable aneuploidy of other chromosomes. It is also possible that one or several common factor(s) is/are involved in both constitutional and sporadic aneuploidy.

Glu274Lys/Gly309Arg mutation of the tissue-nonspecific alkaline phosphatase gene in neonatal hypophosphatasia associated with convulsions.

We describe a patient diagnosed with lethal perinatal hypophosphatasia with a unique clinical presentation of convulsions that responded to vitamin B6. Genomic DNA sequence analysis of the tissue-nonspecific alkaline phosphatase (TNSALP) gene revealed two missense mutations: a G-to-A transition resulting in a Glu to Lys at codon 274 (E274K), and a G-to-C transversion resulting in a Gly to Arg at codon 309 (G309R). The first mutation was maternally transmitted and was previously characterized as a moderate one, whereas the latter was paternally transmitted and has not been previously reported. Phenotype/genotype correlation indicates that G309R is a deleterious mutation that can lead to seizures and a lethal outcome, as was demonstrated in our patient.

Congenital short-bowel and malrotation: clinical presentation and outcome of six affected offspring in three related families.

Newborns with congenital short small bowel associated with malrotation and intestinal dysmotility have a uniformly bad prognosis. However, few long-term survivors have been reported, suggesting that the disorder is not invariably fatal. The majority of cases previously reported were familial. We report on six affected siblings in three related families. The aim of this report is to assess the mode of inheritance, the expression of this disorder, and to point to the correlation between the onset of gastrointestinal symptoms and the outcome.

Replication status as a possible marker for genomic instability in cells originating from genotypes with balanced rearrangements.

Most allelic pairs of DNA replicate synchronously during the S phase of the cell cycle. However, some genes frequently replicate asynchronously, i.e. genes on the X chromosome and imprinted genes. Earlier studies demonstrated an asynchronous pattern of replication in some precancerous and invasive squamous carcinoma of the cervix as well as in multiple myeloma. A high rate of asynchronous pattern was found in: (1) lymphocytes of individuals with solid tumors as well as in other malignancies; (2) amniocytes of genotypes with an extra chromosome 13, 18 and 21; (3) lymphocytes of young mothers of a Down syndrome pregnancy. The asynchronic pattern was not locus specific and was found in all loci analyzed. These findings suggested that the mechanism controlling the temporal order of replication could be altered in cells with a genetic predisposition to cancer or aneuploidy. In this study, we found a higher rate of asynchronous pattern in genotypes carrying inversions 2 and 9 and in balanced heritable translocations (p < 0.01) and an even higher rate in cases with a de-novo balanced translocation. The process of tumorigenesis may begin with a change in cell cycle regulation which includes the duplication, replication and segregation of genetic information. However, it remains unknown whether individuals with balanced chromosome rearrangements are at increased risk of developing cancer later in life.

Prenatal diagnosis for detecting congenital malformations: acceptance among Israeli Arab women.

BACKGROUND: A high rate of consanguineous marriages exists within the Israeli Arab community, with approximately half occurring between first cousins. This contributes towards a high incidence of congenital malformations and autosomal recessive diseases, many of which are detectable at prenatal diagnosis. OBJECTIVES: To assess the levels of both awareness and acceptance regarding prenatal diagnosis and termination of pregnancy among a group of Arab women in order to devise the optimal means of providing genetic counseling and general health services. METHODS: A total of 231 Arab women of childbearing age were interviewed 3 days postpartum to assess their knowledge of prenatal diagnosis and termination of pregnancy, their willingness to undergo prenatal diagnosis, and their opinions on termination of pregnancy in the event of a severely affected fetus. RESULTS: Half the women believed that prenatal testing is not an effective (or accurate) tool for diagnosing an affected fetus. A quarter had poor knowledge on prenatal diagnosis, and a quarter believed that prenatal diagnosis does provide the correct diagnosis. Ninety-five percent said they would agree to undergo prenatal diagnosis; and in the event of a severely affected fetus, 36% said they would agree to a termination of pregnancy, 57% said they would not, and 7% were undecided. CONCLUSIONS: There is a need for special intervention programs, with guidance by health professionals, geneticists and religious authorities, that will inform this population on the increased risk associated with consanguinity, stress the importance and effectiveness of prenatal testing to identify severe congenital malformations, and help them to accept prenatal diagnosis and termination of pregnancy if indicated.

Replication asynchrony increases in women at risk for aneuploid offspring.

We attempted to demonstrate a relation between a loss of replication control, centromere dysfunction, and predisposition to non-disjunction. Couples with a Down syndrome offspring were the high-risk probands. One-color FISH (fluorescent in-situ hybridization) was applied to interphase nuclei (lymphocytes). Replication pattern of two pairs of alleles, RB-1 and 21q22, were studied, and the rate of aneuploidy was estimated using two alpha-satellite probes of chromosomes 8 and 18. Our results suggest the existence of an association between replication timing and the rate of non-disjunction. A higher rate of allele asynchrony and aneuploidy was found in older women and in mothers of a Down syndrome offspring. These findings may reflect a predisposition for meiotic non-disjunction in these women.

Molecular analysis of an extended Palestinian family from Israel with monilethrix.

We describe the molecular analysis of a large three generation Palestinian family segregating for monilethrix. Previous reports have shown that mutations in type-II hair cortex keratin genes, hHb1 and hHb6, are associated with monilethrix. Genetic linkage analysis performed on this family using markers flanking the hHb6 gene exhibited strong evidence for linkage. Sequence analysis revealed a nucleotide substitution of G --> T at nucleotide 1230 resulting in a glutamic acid to aspartic acid amino acid substitution at codon 410, identical to that reported in a French family. The family in our study provides further evidence that mutations of the hHb6 gene are responsible for monilethrix.

Dilemma of trisomy 20 mosaicism detected prenatally: is it an innocent finding?

The clinical significance of mosaicism trisomy 20 detected prenatally following amniocentesis remains uncertain, due to the rarity of liveborn cases with inconsistent clinical findings, the short postnatal follow-up, and failure in evaluating other fetal tissues for the presence of the trisomy. We report on a 15 month-old 46,XX chromosome constitution in white blood cells, while skin fibroblasts demonstrated trisomy 20 mosaicism (54%) by fluorescence in situ hybridization (FISH) analysis. Clinical examination of the baby showed only minor phenotypic signs (bilateral epicanthal folds, delayed closure of fontanel with no other gross anomalies), but demonstrated a considerable developmental delay in gross and fine motor skills along with hypotonicity. This is the second oldest described liveborn with trisomy 20 mosaicism confirmed in skin fibroblasts. This cytogenetic aberration along with her developmental delay suggests that the two findings are related and that aberration affects various fetal tissues and is not confined to extra-embryonic tissue as suggested previously. Yet, an undiagnosed condition may be the cause of the child's developmental delay. Based on this case and following a review of the literature we suggest that when mosaic trisomy 20 is identified in amniocytes, further evaluation is required. Cord blood should be analyzed preferably by FISH. During counseling the parents should be advised of an additional risk, such as developmental delay, even when fetal cord karyotype and detailed ultrasonic scan are normal.

Brain anomalies, retardation of mentality and growth, ectodermal dysplasia, skeletal malformations, Hirschsprung disease, ear deformity and deafness, eye hypoplasia, cleft palate, cryptorchidism, and kidney dysplasia/hypoplasia (BRESEK/BRESHECK): new X-linked syndrome?

Two half brothers (maternally related) had a similar syndrome of microhydrocephaly in both brothers and dilatation of the spinal canal with fusion of thalami in one brother. Primordial growth delay was noted in both brothers, with severe mental retardation in the surviving brother. Both had ectodermal dysplasia with scaling, hyperkeratosis, and generalized alopecia, but normal sweat and sebaceous glands. Skeletal anomalies included hemivertebrae with abnormal segmentation in one and scoliosis with polydactyly in the other. Ears were apparently low set, large, and protruding, with mixed hearing loss in the brother who survived. Eye anomalies included maldevelopment of one eye in Patient 1 and small optic nerves more noticeable on one side in Patient 2. Both had cryptorchidism and dysplastic/hypoplastic kidneys of varying severity that resulted in the early postnatal death of one sib. Manifestations present in only one or the other sib included submucous cleft palate, aganglionosis of the rectum and colon, agenesis of one testicle, and single umbilical artery. This syndrome has not been described previously and may be due to an X-linked mutation. The acronym BRESEK reflects the common findings, whereas BRESHECK denotes all manifestations of both patients: brain, retardation, ectodermal dysplasia, skeletal deformities, Hirschsprung disease, ear/eye anomalies, cleft palate/cryptorchidism, and kidney dysplasia/hypoplasia. In addition to an X-linked mutation, a contiguous gene deletion or maternal mosaicism of an autosomal dominant gene must be considered.

Are all phenotypically-normal Turner syndrome fetuses mosaics?

Cytogenetic and fluorescent in situ hybridization (FISH) studies were performed on several formalin-fixed tissues obtained from four fetuses diagnosed at amniocentesis as 45,XO-Turner syndrome. Three of the four were phenotypically normal and one had malformations. The three phenotypically normal cases were found to have an additional normal cell line, which may explain their ability to survive, at least to the time of pregnancy termination well into the second trimester. The abnormal 45,XO fetus was not found to be mosaic in all of the tissues examined. In 45,XO cases in which no malformation is detected, the possibility of mosaicism should be raised and thus the counselling should be modified accordingly.

Duplication of 7p: further delineation of the phenotype and restriction of the critical region to the distal part of the short arm.

We report on a patient with duplication of 7p15-->pter and review the literature. Patients with partial duplication of the distal 7p, including only the distal segment 7p15-->pter, have a syndrome comparable to that of patients with a larger or complete duplication of 7p. This suggests that the critical region for the dup(7p) phenotype is restricted to 7p15-->pter. The complete clinical phenotype of dup(7)(p15-->pter) includes mental retardation, skull anomalies, large anterior fontanel, cardiovascular defects, joint dislocation and contraction, and gastrointestinal and genital defects. Recognition of the clinical spectrum in patients with a smaller duplication of 7p, and the assignment of this critical region, should prove valuable for accurate counseling, prediction of outcome, and further gene mapping.

Partial monosomy of chromosome 1p36.3: characterization of the critical region and delineation of a syndrome.

We describe 5 patients ranging in age from 3 to 47 years, with karyotypic abnormalities resulting in monosomy for portion of 1p36.3, microcephaly, mental retardation, prominent forehead, deep-set eyes, depressed nasal bridge, flat midface, relative prognathism, and abnormal ears. Four patients have small hands and feet. All exhibited self-abusive behavior. Additional findings in some of the patients include brain anomalies, optic atrophy, hearing loss and skeletal deformities. The breakpoints within chromosome 1 were designated at 1p36.33 (1 case). Thus, the smallest region of deletion overlap is 1p36.33-->1pter. Detection of the abnormal 1 relied on high resolution G-band analysis. Fluorescence in situ hybridization (FISH) utilizing a DNA probe (Oncor D1Z2) containing the repetitive sequences in distal 1p36, confirmed a deletion of one 1 homologue in all 5 cases. The abnormal 1 resulted from a de novo deletion in only one patient. The remaining patients were either confirmed (3 cases) or suspected (1 case) to have unbalanced translocations. Despite the additional genetic imbalance present in these four cases, monosomy of 1p36.33 appears to be responsible for a specific clinical phenotype. Characterization of this phenotype should assist in the clinical diagnosis of this chromosome abnormality.

The molecular basis of ornithine transcarbamylase deficiency: modelling the human enzyme and the effects of mutations.

Human ornithine transcarbamylase is a trimer with 46% amino acid sequence homology to the catalytic subunit of E coli aspartate transcarbamylase. Secondary structure predictions, distributions of hydrophilic and hydrophobic regions, and the pattern of conserved residues suggest that the three dimensional structures of the two proteins are likely to be similar. A three dimensional model of ornithine transcarbamylase was generated from the crystal structure of the catalytic subunit of E coli aspartate transcarbamylase in the holoenzyme, by aligning the sequences, building in gaps, and minimising the energy. The binding sites for carbamyl phosphate in both enzymes are similar and the ornithine binding site in ornithine transcarbamylase appears to be in the same location as the L-aspartate binding site in aspartate transcarbamylase, with negatively charged side chains replaced by positively charged residues. Mutations in the ornithine transcarbamylase gene found in patients with hyperammonaemia of the "neonatal type" are clustered in important structural or functional domains, either in the interior of the protein, at the active site, or at the interchain interface, while mutations found in patients with milder "late onset" disease are located primarily on the surface of the protein. The predicted effects of all known missense mutations and in frame deletions in the ornithine transcarbamylase gene on the structure and function of the mature enzyme are described.