Amal M. Mohamed

Isodicentric Y chromosomes in Egyptian patients with disorders of sex development (DSD).

Isodicentric chromosome formation is the most common structural abnormality of the Y chromosome. As dicentrics are mitotically unstable, they are subsequently lost during cell division resulting in mosaicism with a 45,X cell line. We report on six patients with variable signs of disorders of sex development (DSD) including ambiguous genitalia, short stature, primary amenorrhea, and male infertility with azoospermia. Cytogenetic studies showed the presence of a sex chromosome marker in all patients; associated with a 45,X cell line in five of them. Fluorescence in situ hybridization (FISH) technique was used to determine the structure and the breakage sites of the markers that all proved to be isodicentric Y chromosomes. Three patients, were found to have similar breakpoints: idic Y(qter→ p11.32:: p11.32→ qter), two of them presented with ambiguous genitalia and were found to have ovotesticular DSD, while the third presented with short stature and hypomelanosis of Ito. One female patient presenting with primary amenorrhea, Turner manifestations and ambiguous genitalia revealed the breakpoint: idic Y (pter→q11.1::q11.1→pter). The same breakpoint was detected in a male with azoospermia but in non‐mosaic form. An infant with ambiguous genitalia and mixed gonadal dysgenesis (MGD) had the breakpoint at Yq11.2: idic Y(pter→q11.2::q11.2→pter). SRY signals were detected in all patients. Sequencing of the SRY gene was carried out for three patients with normal results. This study emphasizes the importance of FISH analysis in the diagnosis of patients with DSD as well as the establishment of the relationship between phenotype and karyotype.

Cyclin D1 gene amplification in proliferating haemangioma

Cyclin D1 gene amplification has been reported to promote abnormal endothelial cell proliferation and angiogenesis; these findings constantly present in proliferating haemangiomas. The present study was conducted to evaluate cyclin D1 gene amplification by fluorescence in situ hybridization analysis in tissue biopsies of 22 proliferating haemangiomas from 20 infants. Two significant correlations of cyclin D1 gene amplification with the early onset and the duplication of proliferating haemangiomas have been observed. Moreover, a significant correlation (P≤0.05) has been found between the treatment parameters of proliferating haemangiomas with the amplified versus the normal cyclin D1 gene. Proliferating haemangiomas with the amplified cyclin D1 gene required more frequent flashlamp pulsed dye laser treatment sessions at the maximum dosimetry and more frequent intralesional steroid injections at the maximum dose/injection but treatment outcomes were limited. The more frequent post-treatment complications among proliferating haemangiomas with cyclin D1 gene amplification might be attributable not only to the associated more aggressive natural course, but also to the higher treatment parameters needed for effective treatment. Within the limitations of the present study, cyclin D1 gene amplification was seen for the first time in proliferating haemangiomas. We have found that the amplification of the cyclin D1 gene can predict the more aggressive natural course of proliferating haemangiomas and the limited outcome and higher incidence of complications after non–excision treatment modalities. The present findings reflect the possible usefulness of antisense cyclin D1 to improve the therapeutic outcome of proliferating haemangiomas.

Clinical features of SMARCA2 duplication overlap with Coffin–Siris syndrome

Coffin–Siris syndrome is a rare congenital malformation and intellectual disability syndrome. Mutations in at least seven genes have been identified. Here, we performed copy number analysis in 37 patients with features of CSS in whom no causative mutations were identified by exome sequencing. We identified a patient with a 9p24.3–p22.2 duplication and another patient with the chromosome der(6)t(6;9)(p25;p21)mat. Both patients share a duplicated 15.8‐Mb region containing 46 protein coding genes, including SMARCA2. Dominant negative effects of SMARCA2 mutations may contribute to Nicolaides–Baraitser syndrome. We conclude that their features better resemble Coffin–Siris syndrome, rather than Nicolaides–Baraitser syndrome and that these features likely arise from SMARCA2 over‐dosage. Pure 9p duplications (not caused by unbalanced translocations) are rare. Copy number analysis in patients with features that overlap with Coffin–Siris syndrome is recommended to further determine their genetic aspects.

Intellectual disability secondary to a 16p13 duplication in a 1;16 translocation. Extended phenotype in a four-generation family

We describe a large family from the Gaza Strip presented with multiple congenital anomalies. The proband was presented with intellectual disability and multiple congenital anomalies including cleft palate, low‐set ears, everted upper lip, diaphragmatic hernia, and arthrogryposis. Pedigree analysis showed 19 affected patients over five generations, only 6 were alive and 11 individuals were obligate carriers. The proband had an apparently normal karyotype, although FISH studies showed a derivative chromosome 1 with duplication of 16p13.3 and deletion of the 1p subtelomere. Her father however had a balanced translocation. The seven affected patients had a similar phenotype, one of them died before genetic testing was carried out and the living six patients had the same unbalanced translocation. Array CGH revealed an 8.8 Mb duplication in 16p13 and 200,338 bp deletion in 1p36.3. Accordingly, intellectual disability, hypertelorism, cupped ears, everted upper lip, and limb anomalies were presenting clinical features of the 16p13 duplication syndrome while deep set eyes were perhaps related to the 1p terminal deletion. Prevention of recurrent intellectual disability in this family can be achieved through carrier detection and prenatal genetic diagnosis.

An infertile azoospermic male with 45,X karyotype and a unique complex (Y;14); (Y;22) translocation: cytogenetic and molecular characterization

45,X testicular disorder of sex development (DSD) is a very rare disorder. It usually results from Y/ autosomal translocations or insertion [1–3]. The frequency of Y/ autosome translocations in the general population is generally low occurring in about 1 in 2000 [4]. Y/ autosome (Y;A) translocations are usually balanced and segregate in families with minimal effects on the phenotype [4–6]. On the other hand, unbalanced Y;A reciprocal translocations commonly present with infertility and azoospermia [7–9]. Some patients may present with other presentations related to abnormal sex chromosme dosage as ambiguous genitalia, short stature, and some of the Turner syndrome features . Others may have congeni ta l malformations, and mental subnormality depending on the genes involved in the autosome [2, 3, 10, 11]. Almost all autosomes had been reported, but acrocentrics are the most commonly involved [2, 12–16]. The causative factor of male infertility and spermatogenic failure in Y;A translocation is the disruption of AZF loci by the translocation breakpoint or by a positional effect [17–19]. Another explanation is the occurrence of spermatogenic arrest due to abnormal meiotic pairing with defective X/Y body (sex vesicle) formation and inactivation of autosomal segment and consequently spermatocyte degeneration [13, 20–23].

Unbalanced 14;X Translocation and Pattern of X Inactivation in a Female Patient with Multiple Congenital Anomalies

We report on a female patient who was first evaluated at the age of 6 years with developmental delay, dysmorphic facial features, seizures, and autistic behavior. A brain CT showed complete agenesis of the corpus callosum, and EEG recorded bilateral epileptogenic foci. Karyotype analysis revealed 45,X,psu dic(14;X)(p11;p22). FISH using 14q and Xp subtelomeric probes, combined with a SHOX gene-specific probe, and centromere X and XIST gene analysis revealed ish psu dic(14;X)(D14S1420+; DXYS129-, SHOX-, DXZ1+, XIST+). Array CGH detected a 2-Mb loss at Xp22.33 and a 4.6-Mb gain at Xp22.2p22.12. The deletion contains 34 genes, of which CSF2RA and SHOX are OMIM morbid genes. The duplication also contains some OMIM morbid genes, of which CDKL5, NH5, RPS6KA3, and AP1S2 are the most important. The late replicating chromatin technique was used to detect the pattern of X inactivation in the normal X and in the translocated chromosome. The translocated X was found to be inactive in 70% of the studied blood lymphocytes with patchy extension of inactivation to chromosome 14. In conclusion, the phenotype of the patient may be partially affected by the haploinsufficiency of the genes that are known to escape X inactivation and that lie within the deleted region and by other deleted or duplicated genes on the abnormal X chromosome due to an alternative pattern of X inactivation. The phenotype of the patient was significantly aggravated and complicated by the functional monosomy of some genes on chromosome 14 due to partial spreading of inactivation and silencing of those genes. This case report indicates the importance of structural and functional studies and emphasizes the clinical importance of the follow-up of abnormal microarrays.

Study of Her-2/neu and TOP2A expression in familial versus sporadic breast carcinoma

IntroductionGenetic predisposition is one of the important risk factors that increase the incidence of breast cancer. The Her-2/neu gene is considered the most frequently amplified oncogene and is accompanied with either amplification or deletion of the TOP2A gene with an equal frequency. Aim of the studyThe aim of the study was to compare patients with familial breast cancer (FBC) with those having sporadic breast cancer (SBC) and also to evaluate the combined study on the hormonal status, immunostaining for Her-2/neu, and Her-2/neu and TOP2A copy number alterations by fluorescence in-situ hybridization (FISH). Patients and methodsA total of 22 patients with invasive breast carcinomas were involved: 12 positive for the criteria that define FBC and 10 positive for SBC. Representative sections of formalin-fixed paraffin-embedded blocks were subjected to immunostaining for Her-2/neu (Hercep test), estrogen receptors, and progesterone receptors and also to FISH in order to evaluate TOP2A and Her-2/neu copy number alterations. ResultsFBC tumors were more aggressive than SBC tumors, as assessed by pathological parameters and immunohistochemical markers (lower estrogen receptor and progesterone receptor expression). However, the frequency of Her-2/neu gene amplification and protein overexpression was equal between the two groups. TOP2A gene amplification was observed in 25% of FBC patients compared with 20% of SBC patients. Coamplification of both Her-2/neu and TOP2A genes was observed in 8.3% of FBC patients and in 20% of SBC patients. In contrast, Her-2/neu amplification alone was observed in 25% of FBC patients compared with 10% of SBC patients. Importantly, TOP2A amplification without Her-2/neu gene amplification was observed in 16.7% of FBC patients but was not observed among SBC patients. ConclusionFBC tumors were more aggressive than SBC tumors. This study suggests that TOP2A gene amplification occurs both in FBC and in SBC patients but more frequently among FBC patients. Moreover, TOP2A gene amplification may occur independent of Her-2/neu amplification. We can conclude that this parameter should be determined on a routine basis in breast cancer patients, especially when FBC is suspected. In addition, a combined approach using immunohistochemical analysis and FISH can optimize Her-2/neu testing for breast carcinoma patients, especially among those with immunohistochemistry-equivocal tumors.

Fanconi anemia: studying telomeres and telomerase genes using fluorescent in-situ hybridization technique in Egyptian patients

ObjectiveTo assess telomeric deletion and the telomerase gene copy number in patients with Fanconi anemia (FA) and aplastic anemia. MethodsThe study included 10 patients with aplastic anemia (seven FA and three non-FA) and five healthy individuals. All patients were subjected to diepoxybutane test and fluorescent in-situ hybridization study using all human telomeres and TERC and TERT Gene fluorescent in-situ hybridization probes. ResultsThere was statistically significant difference in telomeric deletion in the patients with FA (P<0.005). Moreover, there was a significant telomeric deletion in patients with aplastic anemia (non-FA) (P<0.016), but there was no significant difference in the deletion between FA and non-FA (P<0.16). In contrast, none of the studied patients exhibited telomerase gene amplification. ConclusionHigh values of telomeric end deletions were detected in our patients (FA and non-FA). Our finding confirms that the increase in the enzyme level is caused by other factors than gene amplification.