Prevalence. Genetic disorders and congenital ab­normalities (also called birth defects) affect between 3 and 5% of the live-births in Europe [1] and in the United States [2]. Congenital anomalies (CA) or birth defects are defined as the presence, at birth, of struc­tural, functional and/or biochemical-molecular de­fects, irrespective of whether they have been detected at that time or not. Congenital malformations (CM), which comprise 60% of all CA, are the major group. A CM is a physical congenital anomaly that is deleteri­ous, i.e., a structural defect perceived as a problem. A typical combination of malformations affecting more than one body part is referred to as a malformation syndrome. Some of the congenital defects are genetic in origin and are referred to as “genetic disorders.”

Congenital malformations are a major cause of fetal, neonatal and infant morbidity and mortality in all industrialized countries [3,4]. They are the fifth leading cause of mortality. Twenty percent of infant deaths are attributed to CM; in developed countries, the percentage has increased over time. The morbid­ity and disability experienced by surviving children also have a major impact on public health [3,4]. Ap­proximately 25% of the number of pediatric hospital admissions and one-third of the total number of pe­diatric hospital days are attributed to different types of CM [5,6]. This is associated with enormous costs for medical care and creates heavy psychological and emotional burdens for the affected individuals and/or their families. Congenital malformations involving the brain comprise the largest group with prevalence of 10/1,000 live births, compared to congenital heart disease (8/1,000), urinary tract (4/1,000) and limb anomalies (1/1,000). The remaining types of CM have a combined prevalence of 6/1,000 live births. Congenital heart anomalies are responsible for 28% of infant deaths related to CM; central nervous sys­tem malformations account for about 12% of infant deaths, while chromosomal and respiratory system abnormalities each account for 15% of infant deaths [1]. Classification. Congenital malformations can be divided into three groups: 1) Lethal if the de­fects (such as anencephaly or hypoplastic left heart syndrome) cause still births (late fetal death), infant death or the pregnancy is terminated after prenatal di­agnosis of fetal defects in more than 50% of cases. 2. Severe if the defects (such as cleft lip or congenital pyloric stenosis) which, without medical intervention, cause handicap or death. 3. Mild if defects (such as congenital dislocation of the hip or undescended tes­tes) require medical intervention but life expectancy is good.

Lethal and severe defects together constitute major congenital abnormalities. Minor anomalies or morphological variants (such as epicanthal folds, ocu­lar hypotelorism, preauricular tags and pits, low-set ears, simian crease, clino- and camptodactyly, partial syndactyly between toes 2 and 3, hydrocele, umbili­cal hernia, sacral dimple, etc.) without serious medi­cal or cosmetic consequences, are excluded from the category of congenital malformations [7]. While mi­nor anomalies in themselves do not greatly affect the child, they can be associated with major anomalies or be indications of certain syndromes [8,9].

Etiological Classification of Congenital Mal­formations [10]. 1) Microscopically visible, unbal­anced chromosome abnormalities. 2) Submicroscopic chromosome abnormalities including microdeletions, uniparental disomy and imprinting mutations. 3) Teratogens and prenatal infections. 4) New dominant mutations. 5) Familial disorders not included as a new dominant mutation. 6) Recognized non familial, non chromosomal syndromes. 7) Isolated anomalies.

For 20% of the CM there seems to be a “multifac­torial” cause, meaning a complex interaction of mul­tiple minor genetic abnormalities with environmental risk factors. Another 10-13% of CM have a purely environmental cause (e.g., infection, illness, medica­tion or drug abuse in the mother). Around 12-25% of CM have a genetic cause. The etiology of CM is not always clear. Some 40-60% of CM have no known cause.[11-13].

Genetic Methods for Determining the Etiol­ogy of Congenital Malformations. The information provided by methods such as fluorescence in situ hy­bridization (FISH), quantitative fluorescence poly­merase chain reaction (QF-PCR), MLPA (multiplex ligation-dependent probe amplification) or classical cytogenetics on etiology of CM is limited, since the majority of these cases do not.detect micro structural genomic imbalances [14]. Array-based techniques have enabled higher resolution screens for genomic imbalances and permit identification of micro struc­tural aberrations between 60 bp and several hundred kilobases in size that are identified only by the size and density of the sequences spotted on the microar­ray. Whole genome screening and detection of novel unbalanced micro structural rearrangements are pos­sible in a single reaction [15].

In the array comparative genomic hybridization (aCGH) method, hybridization of DNA takes place on an array of mapped DNA clones rather than meta­phase chromosomes and leads to “molecular karyo­typing” rather than conventional karyotyping [16,17]. It can be carried out on DNA from single cells, from chorionic villus cells and from amniocytes. Molecular karyotyping has doubled the detection rate of patho­genic chromosomal imbalances by increasing the resolution level from 5 Mb (with conventional karyo­typing) to as low as 100 kb.

In a study of spontaneous miscarriages, aCGH detected all abnormalities previously identified by microscopic karyotype analysis and additional abnor­malities in some 10% of cases [18]. In 98 pregnan­cies (56 amniotic fluid and 42 chorionic villus sample specimens) complete concordance of array results was found for direct and cultured cell analyses in 57 cases tested by both methods [19]. Bar-Shira et al. [20] screened eight patients with multiple CA, mental deficiency and dysmorphic features by aCGH. In two previously undiagnosed cases, they detected chromo­somal micro alterations. Thienpont et al. [21] found by aCGH that 30% of patients with congenital heart defects had imbalances that were not described in phenotypically normal individuals. Menten et al. [22] reported 20% submicroscopic chromosomal imbal­ances, detected by aCGH, in a series of 140 patients with idiopathic multiple congenital malformations and mental retardation having normal karyotypes.

Array CGH appears to be more sensitive for detecting mosaicism than conventional cytogenetic methodologies. One expalanation is that if mosaicism is not suspected on the basis of the clinical findings, the number of metaphases counted may be insuffi­cient to detect the mosaicism. Another explanation is that since the chromosome analysis relies on stimu­lated cells, the aneuploid cells may be under repre­sented in the cell population [23]. In a study of 2,585 samples, chromosomal mosaicism was detected by aCGH in 12 patients, 10 of whom were reported to have normal chromosomes in blood cells [24]. Ballif et al. [25] reported 18 cases of mosaicism detected by aCGH in a routine diagnostic setting. In all cases, FISH confirmed the mosaic chromosome abnormali­ties, showing that the percentage of abnormal cells in unstimulated cultures was, in some cases, different from that found in PHA-stimulated cells. Thus, aCGH based on direct extraction of genomic DNA from un­cultured peripheral blood, may be more likely to de­tect low-level mosaicism than traditional cytogenetic techniques [25]. We have used aCGH to screen for micro structural whole genome copy number changes in five patients with CM and normal karyotypes. Underlying unbal­anced micro structural aberrations were found in two patients with CM, in one a low level mosaicism form of the deletion 18q21.1-q23 and in the other a 1p36 monosomy [26]. It was found that over 12% of the human genome includes submicroscopic benign copy number variable regions [27]. Array CGH has revealed frequent imbal­ances associated with clinical syndromes, but also a large number of copy number variations (CNVs) ­large segments of DNA, ranging in size from thou­sands to millions of DNA bases with variations in the copy number. Some of these variations may represent risk factors for particular clinical anomalies. A CNV is operationally defined as a DNA segment, longer than 1 kb, with a variable copy number compared with a reference genome. This definition may not be useful in deciding the clinical impact of certain genomic im­balances. Copy number variations may be categorized into those that are likely to be benign (polymorphic), those that are likely to be pathogenic and those of un­known clinical significance [28-30].

Factors that influence the risk contribution of a CNV: if the genomic imbalance is found in the affect­ed individual and in a healthy parent, it is more likely to be a benign CNV. To determine which imbalances are pathogenic, one could simply tabulate all those ob­served in an individual’s aCGH test, disregard CNVs found in normal individuals and consider the remain­ing copy number changes as potentially pathogenic. The potential clinical relevance of a CNV increases in proportion to the number of genes within the region of genomic imbalance. As it is generally thought that duplications are better tolerated in the genome than deletions, deletion CNVs have a higher likelihood of being pathogenic.

Of 14 patients with a syndrome of aplasia of the Müllerian ducts, urinary tract anomalies, cardiac and skeletal defects, hearing impairment, and mental re­tardation, four had cryptic genomic alterations; all of which were independently ascertained and did not overlap. There were two duplications in one and three different deletions in the other three patients [31].

Of 49 fetuses with multiple malformation and normal karyotypes investigated by aCGH, eight had genomic rearrangements (16.3%). These included: subtelomeric deletions, interstitial deletions, submi­croscopic duplications and multiplex genomic imbal­ance [32].

Array CGH is often used as a primary genetic screening method for diagnosis and research. The technique can detect pathogenic submicroscopic chro­mosomal imbalances in patients with developmental disorders. Most patients carry different chromosomal anomalies, which may be spread across the whole genome. These imbalances locate genes that are in­volved in human development. This is important for phenotype/genotype correlations and for the identi­fication of genes. Since most imbalances encompass regions harboring multiple genes, the challenge is: 1) to identify those genes responsible for the specific phenotype, and 2) to disentangle the role of the dif­ferent genes located in an imbalanced region. The high resolution of aCGH makes it a basic research 1 kb, with a variable copy number compared with a reference genome. This definition may not be useful in deciding the clinical impact of certain genomic im­balances. Copy number variations may be categorized into those that are likely to be benign (polymorphic), those that are likely to be pathogenic and those of un­known clinical significance [28-30].

Factors that influence the risk contribution of a CNV: if the genomic imbalance is found in the affect­ed individual and in a healthy parent, it is more likely to be a benign CNV. To determine which imbalances are pathogenic, one could simply tabulate all those ob­served in an individual’s aCGH test, disregard CNVs found in normal individuals and consider the remain­ing copy number changes as potentially pathogenic. The potential clinical relevance of a CNV increases in proportion to the number of genes within the region of genomic imbalance. As it is generally thought that duplications are better tolerated in the genome than deletions, deletion CNVs have a higher likelihood of being pathogenic.

Of 14 patients with a syndrome of aplasia of the Müllerian ducts, urinary tract anomalies, cardiac and skeletal defects, hearing impairment, and mental re­tardation, four had cryptic genomic alterations; all of which were independently ascertained and did not overlap. There were two duplications in one and three different deletions in the other three patients [31].

Of 49 fetuses with multiple malformation and normal karyotypes investigated by aCGH, eight had genomic rearrangements (16.3%). These included: subtelomeric deletions, interstitial deletions, submi­croscopic duplications and multiplex genomic imbal­ance [32].

Array CGH is often used as a primary genetic screening method for diagnosis and research. The technique can detect pathogenic submicroscopic chro­mosomal imbalances in patients with developmental disorders. Most patients carry different chromosomal anomalies, which may be spread across the whole genome. These imbalances locate genes that are in­volved in human development. This is important for phenotype/genotype correlations and for the identi­fication of genes. Since most imbalances encompass regions harboring multiple genes, the challenge is: 1) to identify those genes responsible for the specific phenotype, and 2) to disentangle the role of the dif­ferent genes located in an imbalanced region. The high resolution of aCGH makes it a basic research instrument. It helps in defining and refining the criti­cal regions for a disease or a phenotype. This has led to a dramatic increase in gene identification through molecular karyotyping; it is likely that the function of many more genes will be identified in this way.

The ascertainment of unbalanced genomic mi­cro aberrations through aCGH in syndromic patients may lead to the description of new syndromes and to the recognition of a broader spectrum of features for already described syndromes [22,28,33-38]. Array CGH is a rapid and reproducible procedure that pro­vides reliable results in 5 days. It may develop into an excellent tool also for prenatal genetic diagnosis and holds promise for more accurate genetic counseling and reproductive risk assessment.

Hereditary diseases and CAoccupy the third place in the morbidity structure of newborns in the neonatal period (11.4%). Congenital anomalies are ranked first in the infant mortality structure (40% of cases). In all industrialized countries, large scale programs for prevention of congenital anomalies have been devel­oped.

Prevention approaches are often classified into three levels: 1) Primary prevention: avoiding the cause(s) of congenital abnormalities, e.g., rubella vaccination or periconceptional folic acid/multivita­min supplementation. 2) Secondary prevention: early detection followed by effective early treatment, e.g., congenital dislocation of the hip, also undescended testes. Previously, selective abortion/termination of pregnancy following the prenatal diagnosis of se­vere fetal defects, was also referred to as secondary prevention. Recently, the World Health Organization and other international bodies have excluded this ap­proach from the term “prevention.” 3) Tertiary pre­vention: complete recovery of CM by early surgical intervention without residual defects or minimal after effects. Tertiary prevention allows the achievement of complete recovery in 33.5% of cases with CM.

The major proportion of CM (85.3%) are prevent­able; however, no single strategy for their prevention exists [7]. For these and for other reasons, prenatal di­agnosis has long been recognized as an essential facet in the clinical management of the pregnancy itself, as well as a critical step toward the detection, preven­tion, and, eventually, treatment of genetic disorders. Array CGH offers new possibilities for prevention. It makes possible the genetic analysis of single cells; thus, it might give future opportunities for aneuploidy screening and detection of unbalanced translocations in preimplantation embryos [39].