Cytogenetic Analysis of 5572 Patients Referred for Suspected Chromosomal Abnormalities in Morocco

Introduction

Chromosomal abnormalities cause diverse functional problems to various organs and are frequently accompanied by mental retardation, the reason for which, this category of patients need a continuous familial and social support. Even though the economic status and strategies for prenatal diagnosis have progressed recently, management of patients with chromosome abnormalities still remains an important medical problem (Kim et al., 1999). Chromosomal abnormalities are now known to account for a large proportion of spontaneous pregnancy loss and childhood disability, and can also contribute to the genesis of a significant proportion of malignancy in both childhood and adult life as a consequence of acquired somatic chromosomal aberrations (Mueller and Young, 1995).

Chromosomal abnormalities have a great impact during the fetal life because of their high frequency; they represent a major cause of fetal loss. The frequency of various chromosomal abnormalities is quite different in neonates (0.7%) as compared with abortuses (about 50%) since some aneuploidies are lethal in utero (Lauritsen, 1976; Mokhtar, 1997). The major autosomal abnormalities share a number of phenotypic features that are not distinctive or specific including mental retardation, cardiac malformation, and growth deficiency. While there is variability within every cytogenetic syndrome, neonatal death and serious congenital malformations are frequent manifestations. Most of the specific cytogenetic syndromes have a constellation of features that distinguish them and allow the clinician to suspect the condition.

In this study, we determined the commonest causes for requesting cytogenetic analysis of the Department of Medical Genetics of the Moroccan National Institute of Health. In addition, we calculated the prevalence of chromosomal aberration in a referred population for cytogenetic investigation in our department and compared these figures with those reported recently in similar studies. We hope that awareness of these frequencies will help clinicians working in Morocco and surrounding countries to determine the priority for requesting a cytogenetic analysis in individual cases. It should also help to recognize the commonest presentation of the prevalent chromosomal abnormalities in the area.

Patients and Methods

Five thousand five hundred and seventy-two patients (cancer patients excluded) referred to the Moroccan Department of Medical Genetics between 1993 and 2010 were investigated. The study included patients with various phenotypic abnormalities, such as multiple congenital anomalies (300 cases), short stature (529 cases), dysmorphic features with developmental delay (508 cases), unclassified mental retardation (135 cases), Down syndrome (1260 cases), abnormality in sexual differentiation (226 cases), infertility (269 cases), recurrent miscarriage (703 cases), amenorrhea (219 cases), Klinefelter syndrome (118 cases), chromosome breakage syndromes (84 cases), screening of sibships with a known member with abnormal karyotype (82 cases), and miscellaneous (679).

All patients were subjected to a full genetic analysis; complete genetic examination and pedigree construction was done to exclude known nonchromosomal causes of the anomaly.

Cytogenetic analysis was carried out for all the patients. The study included peripheral lymphocyte culture by a standard method using a reverse banding technique (RHG banding). Other appropriate banding techniques such as G-banding technique using trypsin, centromere-banding (C-banding), and nucleolar organizing region staining were employed when needed. About 0.4-0.8 mL of peripheral blood was incubated in complete lymphocyte culture medium for 72 h. Metaphases were harvested by adding karyomax colcemid solution for 50 min followed by hypotonic KCl (0.075 M) treatment for 20 min and fixation using standard 3:1 methanol and acetic acid fixative (Dutrillaux and Couturier, 1981). At least 11 metaphases were scored for each patient. The best metaphases were karyotyped and the total chromosome count was usually determined in 11 cells. If mosaicism was suspected, then 22 or more cell counts were carried out for documentation for abnormal cases.

A high-resolution analysis was done by synchronization using thymidine solution (15 mg/mL) for 16 h before harvesting (Dutrillaux and Couturier, 1981). For fragile X analysis we examined cells cultured in two types of medium: low folate medium and methotrexate-containing medium for the last 24 h of culture.

We used two cytogenetic methods for diagnosis of chromosome breakage syndromes such as Fanconi anemia: evaluation of spontaneous breakage in standard preparations (untreated cultures) and induced chromosome breakage (Seyschad et al., 1995). A minimum of 50 metaphases from each specimen were examined.

Fluorescent in situ hybridization (FISH) was used if required. For the nomenclature of human chromosomes, the Paris conference Paris supplement (Prieur et al., 1973) and the international system for human cytogenetic nomenclature were adopted (ISCN, 2005). Whenever translocations or unusual karyotypes were found, blood samples were collected from both parents and their chromosomes were also studied.

The relative frequency of each diagnostic group was calculated; the percentage of abnormal cases and the distribution of the numerical and structural abnormalities were determined in each group. The frequencies were compared with similar studies.

Results

The most common clinical reasons for performing cytogenetic investigations were suspected Down syndrome (22.61%), recurrent miscarriage (12.62%), short stature (9.49%), dysmorphic features with developmental delay (9.11%), multiple congenital anomalies (5.38%), abnormality in sexual differentiation (4.05%), infertility (4.82%), amenorrhea (3.93%), unclassified mental retardation (2.42%), suspected Klinefelter syndrome (2.12%), chromosome breakage syndromes (1.5%), screening of sibships with a known member with abnormal karyotype (1.47%), and miscellaneous (12.18%).

Of the 5572 cases evaluated, 1504 (27%) showed chromosomal aberrations (Table 1) among which autosome abnormalities were found in 1233 cases (22.13%) (Table 2), while only 220 (3.95%) involved gonosomes (Table 3).

One thousand one hundred and forty patients had autosomal trisomy. Trisomy 21 (Down syndrome) was detected in 1095 cases (96.56% of autosomal trisomy) forming the majority (Table 2).

Of the structural chromosome aberrations of the autosome, reciprocal and Robertsonian translocation was the most common (69 cases), deletion (27 cases), microrearrangements (20 cases), ring chromosome (8 cases), marker chromosome (7), and additions (7).

Two hundred and twenty patients had sex chromosome aberrations (Table 3). Turner's syndrome was confirmed in 122 female patients. The homogeneous monosomy 45,X was the most frequent anomaly (53 cases) and the other abnormalities were either mosaics (28 cases) or structural X abnormalities, including long-arm isochromosome (35 cases), short-arm deletion (2 cases), long-arm deletion (1 case), and X ring chromosome (3 cases). Of the 28 mosaics 8 had a Y chromosome component (46,XY/45,X).

Klinefelter syndrome was detected in 38 male patients. The homogeneous trisomy 47,XXY was the most frequent anomaly (30 cases).

Two hundred and twenty-six cases referred for chromosomal sex assignment were received and in all the cases chromosomal sex was determined. In disorders of sexual differentiation, chromosomal analysis detected 178 cases with concordance between the chromosomal sex and the appearance of the external genitalia. In contrast, 48 cases had discordance between the chromosomal sex and the phenotype. We identified fragile sites on X chromosomes in two boys of all subjects for whom special folate-deficient culture media were used.

Spontaneous chromosome breakage was detected in 19 cases (suspected anemic syndrome). Induced chromosome breakage (after exposure to Mitomycin C) confirmed diagnosis of Fanconi anemia in two patients.

Discussion

In the current study, we detected a rate of chromosomal abnormalities (27%) in a total of 5572 cases. Similar frequencies have been reported previously in other studies, including 31.7% (Al-Awadi et al., 1985), 29.3% (Duarte et al., 2004), 28.6% (Stanos et al., 2000), and 28.3% (Goud et al., 2005), while lower frequencies of 17.5% (Kim et al., 1999) and 3.8% (Kumar et al., 2001) have also been reported. This wide range in frequency of chromosomal aberrations (Table 4) (Singh, 1977; Verma and Dosik, 1980; Navsaria et al., 1993; Mokhtar, 1997; Kim et al., 1999; Duarte et al., 2004; Goud et al., 2005; Balkan et al., 2010) could reflect variations in the criteria for inclusion of the patients in cytogenetic investigations and in the cytogenetic methods used.

In studies based on a referred population with phenotypic abnormalities, such as in the present work, autosomal abnormalities (22.13%) are much higher than those of the gonosomes (3.95%). This percentage is in agreement with other surveys and is mainly due to the fact that a sex chromosome imbalance has a much less deleterious effect on the phenotype than autosomal aneuploidy (Mokhtar, 1997).

Down syndrome is the most common autosomal syndrome and shows a strong association between increasing incidence and advancing maternal age (Mueller and Young, 1995). The frequency of Down syndrome in patients with abnormal chromosomes in the current study was 72.80%. This value is similar to that of other surveys (Mokhtar, 1997). This could be attributed to its easy detection at the clinical level. The distribution of different anomalies associated with Down syndrome in the current study is very similar to earlier reports. Regular trisomy 21 was 96%, in agreement with other surveys, which range from 84.6% to 95.0% (McKinlay Gardner and Sutherland, 1996). We found the frequency of translocations to be 3.1%, a value similar to a study performed in the Sultanate of Oman (Goud et al., 2005) and lower than other studies (Wright et al., 1967; Mikkelsen et al., 1990; Mokhtar, 1997). Only 1.42% of patients with Down syndrome in the current study had mosaicism. The co-occurrence of two numerical chromosomal abnormalities in the same individual (double aneuploidy) is relatively rare and its clinical presentation is variable depending on the predominating aneuploidy or a combination effect of both (Zaki et al., 2005). In this study we found two cases with mosaic double trisomy involving chromosome 21 and sex chromosomes. The first one had double trisomy, Down-Klinefelter, (47,XY,+21/48,XYY,+21) and the second patient had mosaic with 2 cell lines (47,XY,+21/48,XXY,+21) (Ratbi et al., 2006). Trisomy 18 and Trisomy 13 formed the second and third largest trisomy group in the current study.

Turner's syndrome is a total or partial X chromosome monosomy. It affects one in 2500 live-birth in women. Our study showed homogeneous monosomy 45,X as the most frequent anomaly (53 cases). Other abnormalities were either mosaics or structural X abnormalities, including long-arm isochromosome (35 cases), short-arm deletion (2 cases), long-arm deletion (1 case), and X ring chromosome (3 cases). Those results conform to those reported in similar studies (Sybert and McCauley, 2004).

For structural autosomal chromosome aberrations, translocation was the most common (69 cases); deletion (27 cases), and marker chromosome and ring chromosome (14 cases) followed in order of frequency. The identification of small structural abnormalities help not only in diagnosis and counseling but also in gene mapping, like the case reported in a Moroccan family with a balanced translocation t(3;10)(p24;q32) co-segregating with autosomic recessive microcephaly, micropolygyria, and corpus callosum agenesia (Baala et al., 2007). In addition, some chromosomal abnormalities have scientific interest as in the case of unexpected fertility in a Moroccan man with der (22; 22) (q10; 10q), which allowed identification of a new case of paternal uniparental disomy 22 (Ouldim et al., 2004).

The most common supernumerary marker chromosomes (SMCs) are known to be SMCs originating from chromosome 15, in the form of inv dup (15), constituting approximately half of the cases in population studies (Buckton et al., 1985; Woo et al., 2003). Besides the identification of marker chromosomes, clinical correlation, parental study, and consideration of other possibilities are important in genetic counseling of patients with marker chromosomes. In present series four marker chromosomes (de novo) were found and identified using FISH. We also found two bisatellited marker chromosomes (50%) originating from chromosome 15 (inv dup15). One SMC originated from chromosome 18 (iso18p) but the origin of one SMC could not be identified.

This study also indicates that karyotyping still plays an important role in gender assignment of infants with ambiguous genitalia.

Through a combination of high-resolution prometaphase banding and FISH, an increasing number of syndromes are being shown to be due to microdeletions. Some of these microdeletions involve loss of only a few genes at closely adjacent loci, resulting in what is known as contiguous gene syndromes (Mueller and Young, 1995). In this study we found 15 cases of William's syndrome and 5 cases of DiGeorge syndrome. These results are under estimation since FISH technology was only recently introduced into our laboratory.

Chromosome breakage syndromes are characterized by an excess of chromosomal breaks and gaps as well as an increased susceptibility to neoplasia. The Department of Medical Genetics has focused on cytogenetic diagnosis of Fanconi anemia.

Some variant karyotypes including pericentric inversion, hetechromatin, and large Y chromosome were identified in our series (39 cases). These karyotypes were generally considered as normal variants without phenotypic effects on the individuals carrying these aberrations.

Conclusion

Cytogenetic techniques are very important for the correct identification of a variety of syndromes. This is the first report on cytogenetic testing in Morocco. This type of study provides a basis for determining the risks of recurrence and for deciding on clinical treatment and genetic counseling.