The role of candidate-gene CNTNAP2 in childhood apraxia of speech and specific language impairment.

Childhood apraxia of speech (CAS) is a debilitating pediatric speech disorder characterized by varying symptom profiles, comorbid deficits, and limited response to intervention. Specific Language Impairment (SLI) is an inherited pediatric language disorder characterized by delayed and/or disordered oral language skills including impaired semantics, syntax, and discourse. To date, the genes associated with CAS and SLI are not fully characterized. In the current study, we evaluated behavioral and genetic profiles of seven children with CAS and eight children with SLI, while ensuring all children were free of comorbid impairments. Deletions within CNTNAP2 were found in two children with CAS but not in any of the children with SLI. These children exhibited average to high performance on language and word reading assessments in spite of poor articulation scores. These findings suggest that genetic variation within CNTNAP2 may be related to speech production deficits.

Variable expressivity of a novel mutation in the SCN1A gene leading to an autosomal dominant seizure disorder.

Mutations in the SCN1A gene can cause a variety of dominantly inherited epilepsy syndromes. Severe phenotypes usually result from loss of function mutations, whereas missense mutations cause a milder phenotype by altering the sodium channel activity. We report on a novel missense variant (p.Val1379Leu) in the SCN1A gene segregating in an autosomal dominant pattern in a family exhibiting a variable epilepsy phenotype ranging from generalized epilepsy with febrile seizures during infancy to a well controlled seizure disorder in adulthood. This report supports the importance of SCN1A mutation analysis in families in which seizure disorders segregate in an autosomal dominant fashion.

Rapid aneuploidy screening with fluorescence in-situ hybridisation: is it a sufficiently robust stand-alone test for prenatal diagnosis?

OBJECTIVES: To assess the clinical utility of fluorescence in-situ hybridisation with chromosomes 13, 18, 21, X and Y as a stand-alone test in detecting chromosomal abnormalities, and the types of chromosomal abnormalities missed. DESIGN: Retrospective analysis. SETTING: A restructured Government hospital in Singapore and an academic hospital in the United States. PARTICIPANTS: Cytogenetic data of prenatal specimens and results of fluorescence in-situ hybridisation of 5883 patients performed between January 2000 and August 2007 were reviewed. RESULTS: Fluorescence in-situ hybridisation detected 558 (9.5%) patients with chromosomal abnormalities. Abnormal ultrasounds (70%) and maternal serum screens (21%) were the most indicative of chromosomal abnormalities. When comparing fluorescence in-situ hybridisation data with karyotype results for the five chromosomes of interest, the sensitivity and specificity were 99.3% and 99.9%, respectively. When comparing fluorescence in-situ hybridisation data with karyotype results for all chromosomes, the sensitivity decreased to 86.8%, whereas the specificity remained at 99.9%. Of 643 cases with karyotype abnormalities, 85 were fluorescence in-situ hybridisation-negative (false negative rate, 13.2%), which included structural rearrangements, chromosome mosaicism, and other trisomies. Despite abnormal ultrasound indications, fluorescence in-situ hybridisation missed 32 cases which included structural rearrangements, mosaicisms, and other trisomies. CONCLUSION: This study does not support fluorescence in-situ hybridisation as a stand-alone test. Institutions supporting fluorescence in-situ hybridisation as a stand-alone test must seriously consider the risks of a missed diagnosis.

Specific cytogenetic abnormalities are associated with a significantly inferior outcome in children and adolescents with mature B-cell non-Hodgkin's lymphoma: results of the FAB/LMB 96 international study.

Clinical studies showed that advanced stage, high LDH, poor response to reduction therapy and combined bone marrow and central nervous system disease are significantly associated with a decreased event-free survival (EFS) in pediatric mature B-cell non-Hodgkin's lymphoma (B-NHL) treated on FAB/LMB96. Although rearranged MYC/8q24 (R8q24) is characteristic of Burkitt lymphoma (BL), little information is available on other cytogenetic abnormalities and their prognostic importance. We performed an international review of 238 abnormal karyotypes in childhood mature B-NHL treated on FAB/LMB96: 76% BL, 8% Burkitt-like lymphoma, 13% diffuse large B-cell lymphoma (DLBCL). The main BL R8q24-associated chromosomal aberrations were +1q (29%), +7q and del(13q) (14% each). The DLBCL appeared heterogeneous and more complex. Incidence of R8q24 (34%) was higher than reported in adult DLBCL. The prognostic value of cytogenetic abnormalities on EFS was studied by Cox model controlling for the known risk factors: R8q24, +7q and del(13q) were independently associated with a significant inferior EFS (hazard ratio: 6.1 (P=0.030), 2.5 (P=0.015) and 4.0 (P=0.0003), respectively). The adverse prognosis of R8q24 was observed only in DLBCL, whereas del(13q) and +7q had a similar effect in DLBCL and BL. These results emphasize the significant biological heterogeneity and the development of cytogenetic risk-adapted therapy in childhood mature B-NHL.

Producing primate embryonic stem cells by somatic cell nuclear transfer.

Derivation of embryonic stem (ES) cells genetically identical to a patient by somatic cell nuclear transfer (SCNT) holds the potential to cure or alleviate the symptoms of many degenerative diseases while circumventing concerns regarding rejection by the host immune system. However, the concept has only been achieved in the mouse, whereas inefficient reprogramming and poor embryonic development characterizes the results obtained in primates. Here, we used a modified SCNT approach to produce rhesus macaque blastocysts from adult skin fibroblasts, and successfully isolated two ES cell lines from these embryos. DNA analysis confirmed that nuclear DNA was identical to donor somatic cells and that mitochondrial DNA originated from oocytes. Both cell lines exhibited normal ES cell morphology, expressed key stem-cell markers, were transcriptionally similar to control ES cells and differentiated into multiple cell types in vitro and in vivo. Our results represent successful nuclear reprogramming of adult somatic cells into pluripotent ES cells and demonstrate proof-of-concept for therapeutic cloning in primates.

Distinctive patterns of BCL6 molecular alterations and their functional consequences in different subgroups of diffuse large B-cell lymphoma.

Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) has revealed biologically and prognostically distinct subgroups: germinal center B-cell-like (GCB), activated B-cell-like (ABC) and primary mediastinal (PM) DLBCL. The BCL6 gene is often translocated and/or mutated in DLBCL. Therefore, we examined the BCL6 molecular alterations in these DLBCL subgroups, and their impact on BCL6 expression and BCL6 target gene repression. BCL6 translocations at the major breakpoint region (MBR) were detected in 25 (18.8%) of 133 DLBCL cases, with a higher frequency in the PM (33%) and ABC (24%) subgroups than in the GCB (10%) subgroup. Translocations at the alternative breakpoint region (ABR) were detected in five (6.4%) of 78 DLBCL cases, with three cases in ABC and one case each in the GCB and the unclassifiable subgroups. The translocated cases involved IgH and non-IgH partners in about equal frequency and were not associated with different levels of BCL6 mRNA and protein expression. BCL6 mutations were detected in 61% of DLBCL cases, with a significantly higher frequency in the GCB and PM subgroups (>70%) than in the ABC subgroup (44%). Exon-1 mutations were mostly observed in the GCB subgroup. The repression of known BCL6 target genes correlated with the level of BCL6 mRNA and protein expression in GCB and ABC subgroups but not with BCL6 translocation and intronic mutations. No clear inverse correlation between BCL6 expression and p53 expression was observed. Patients with higher BCL6 mRNA or protein expression had a significantly better overall survival. The biological role of BCL6 in translocated cases where repression of known target genes is not demonstrated is intriguing and warrants further investigation.

Inactivation of the E3/LAPTm5 gene by chromosomal rearrangement and DNA methylation in human multiple myeloma.

Chromosomal band 1p34-36 is a commonly rearranged locus in many types of cancers. We cloned the breakpoint region of a chromosomal translocation, t(1;14)(p34;q32), found in the human multiple myeloma (MM) cell line, ODA. This rearrangement occurred between the nearby switch region of the immunoglobulin heavy chain (IgH) gene (Sgamma3) at 14q32 and the first intron of the human retinoic acid-inducible E3 protein (E3)/lysosome-associated protein, transmembrane-5 (LAPTm5) gene at the 1p34 locus. Consequently, the E3 gene, which is a hematopoietic cell-specific transcript induced by retinoic acid and located at the rearranged allele, was interrupted within its coding region and was not expressed in the ODA cell line in spite of the other allele still being intact. The expression derived from the remaining intact allele in ODA cells was silenced by DNA methylation at sequences within the first intron around a GC-rich EagI site. Interestingly, the silenced expression of E3 mRNA due to DNA methylation of intron 1 sequences was frequently encountered in MM cells [6/10 (60%) of MM cell lines tested], while E3 is expressed in normal plasma cells and in most other hematopoietic cell lines including those of B-cell lineage. Thus, as the E3 protein has been suggested to be involved in cellular differentiation and apoptotic pathways in certain cell types, our results suggest that loss of E3 gene expression might be a crucial event during the progression of human MM.

Familial multiple myeloma: a family study and review of the literature.

BACKGROUND: The etiology of multiple myeloma (MM) remains obscure, although reports of familial clustering have implicated both a host susceptibility factor and environmental effects. Here we describe the medical histories of members of a family prone to MM. METHODS: We developed a pedigree for an MM-prone family by using information obtained from a questionnaire. Protein immunoelectrophoresis of serum and urine from the proband and from 19 family members was performed to detect monoclonal immunoproteins. Peripheral blood obtained from the proband and from five relatives was subjected to standard cytogenetic studies to detect constitutional chromosomal abnormalities. Multifluor-fluorescence in situ hybridization (M-FISH) and standard FISH studies were performed on peripheral blood from the proband and from two other affected living relatives to determine their karyotypes and to detect clonal chromosomal abnormalities frequently seen in patients with MM. RESULTS: Within this family, a sibship of seven included three individuals (including the proband) with histologically verified MM and two individuals with a monoclonal gammopathy of unknown significance (MGUS), as determined by immunoelectrophoresis of serum and urine. This family also had members with acute lymphocytic leukemia, malignant melanoma, and prostate cancer. In the family members tested, we detected no constitutional chromosomal abnormality. None of the three individuals analyzed by FISH had a deletion of the retinoblastoma (Rb-1) locus, which is frequently deleted in patients with MM, and only one (the proband) had a translocation involving chromosomes 11 and 14, a clonal abnormality commonly seen in MM. CONCLUSION: The study of familial MM may provide insights into the pathogenesis and, ultimately, the control and prevention of MM and related disorders.

Holoprosencephaly associated with an apparent isolated 2q37.1-->2q37.3 deletion.

A female infant survived 5(1/2) hours after delivery at 33 weeks gestation. Autopsy showed a lobar variant of holoprosencephaly (HPE). Cytogenetic analysis revealed a 2q37.1-->2q37.3 deletion. This case represents the fourth reported case of HPE associated with partial monosomy 2q37 and the first with an apparent isolated 2q37 deletion. Chromosome segment 2q37.1-->2q37.3 may harbor yet another locus important in forebrain development, which, when disrupted, can lead to brain malformations within the HPE spectrum.

Full hematopoietic engraftment after allogeneic bone marrow transplantation without cytoreduction in a child with severe combined immunodeficiency.

Bone marrow transplantation (BMT) for severe combined immunodeficiency (SCID) with human leukocyte antigen (HLA)-identical sibling donors but no pretransplantation cytoreduction results in T-lymphocyte engraftment and correction of immune dysfunction but not in full hematopoietic engraftment. A case of a 17-month-old girl with adenosine deaminase (ADA) deficiency SCID in whom full hematopoietic engraftment developed after BMT from her HLA-identical sister is reported. No myeloablative or immunosuppressive therapy or graft-versus-host disease (GVHD) prophylaxis was given. Mild acute and chronic GVHD developed, her B- and T-cell functions became reconstituted, and she is well almost 11 years after BMT. After BMT, repeated studies demonstrated: (1) Loss of a recipient-specific chromosomal marker in peripheral blood leukocytes (PBLs) and bone marrow, (2) conversion of recipient red blood cell antigens to donor type, (3) conversion of recipient T-cell, B-cell, and granulocyte lineages to donor origin by DNA analysis, and (4) increased ADA activity and metabolic correction in red blood cells and PBLs.

Overview of genetics and role of the pediatric physical therapist in the diagnostic process.

PURPOSE: The purpose of this report is to enhance awareness about the pathogenesis of various genetic diseases and syndromes that physical therapists frequently encounter. The intent is to summarize various aspects of genetic diseases and how they relate to the role of the physical therapist. SUMMARY OF KEY POINTS: Specifically, the origination and development of some of the genetic diseases physical therapists are likely to encounter are discussed, as are resources available to physical therapists and other professionals for further understanding specific genetic diseases. Clinical features of specific genetic diseases can be found in the references provided. RECOMMENDATIONS: Physical therapists can play a primary role in the intricate management of genetic disease in a particular patient and address family and social issues associated with these conditions. It is recommended that the physical therapist work closely with genetics professionals so that a mutual and further understanding can be developed for the benefit of all patients with genetic diseases.

Mantle cell lymphoma. A clinicopathologic study of 68 cases from the Nebraska Lymphoma Study Group.

Although mantle cell lymphoma (MCL) is considered a distinctive disease entity within non-Hodgkin's lymphoma (NHL), the cytology and growth pattern of MCL can be quite variable and the clinical significance of these features is unclear. Also, the role of anthracyclines in the management of MCL is unclear. Therefore, we examined our experience with MCL in an effort to clarify these important issues. We identified 68 patients with MCL who were evaluated clinically and treated by the Nebraska Lymphoma Study Group. Treatment consisted of combination chemotherapy containing an anthracycline in 76% of the patients. The cases were grouped by blastic or lymphocytic cytology, and the latter were divided by growth pattern into nodular (or mantle-zone) and diffuse types. The clinical and pathological variables were then evaluated for their prognostic value. The median overall survival (OS) and failure-free survival (FFS) for the entire group were 38 months and 12 months, respectively, and there was no survival advantage for those who received an anthracycline. The cases were grouped as follows: blastic type, 26%; nodular lymphocytic type, 44%; and diffuse lymphocytic type, 30%. Both the cytology and pattern of growth were predictive of OS and FFS. The median OS was as follows: blastic type, 55 months; nodular lymphocytic type, 50 months; and diffuse lymphocytic type, 16 months (P = 0.0038). The clinical features that predicted for a shorter survival included bone marrow involvement, advanced stage disease, B symptoms, a poor performance score, and the International Prognostic Index. We conclude that new therapeutic approaches, with the patients stratified by histologic type and clinical prognostic factors, are clearly needed for MCL.

Clinical significance of the t(14;18) and BCL2 overexpression in follicular large cell lymphoma.

Follicular large cell lymphoma (FLCL) is an aggressive disease that responds to anthracycline-containing chemotherapy much like diffuse large B-cell lymphoma (DLBCL). Since the t(14;18) and/or bcl2 protein expression are less common in FLCL than in its low-grade counterparts, we sought to determine whether these features were predictive of survival as in DLBCL. We studied 50 patients with FLCL who were treated with curative intent. The t(14;18) was found by cytogenetic analysis in 56% of the patients and bcl2 protein was expressed by the tumor cells in 73%, but neither was predictive of survival. However, abnormalities of chromosome 17p and the presence of trisomy 21 were adverse predictors of survival, as were a number of clinical features. We conclude that neither the absence of the t(14;18) nor the lack of bcl2 expression explain the good response of a subset of patients with FLCL to curative therapy.

Interleukin-1beta upregulates MMP-9 expression in stromal cells of human giant cell tumor of bone.

Giant cell tumor (GCT) of bone is a progressive, potentially malignant process that destroys skeletal tissue. It consists of multinucleated giant cells, which are hypothesized to be derived from a monocyte/macrophage lineage and mononuclear stromal cells, and the precise relationship of these cells is not fully understood. Recently, we demonstrated that the production of matrix metalloproteinase-9 (MMP-9) in GCT stromal cells is regulated by certain factor(s) secreted by the multinucleated giant cells. In the present study, we evaluated for the presence of interleukin-1beta (IL-1beta) and attempted to establish its possible role for the induction of MMP-9 in GCT stromal cells. Using enzyme-linked immunosorbent assay (ELISA), we have demonstrated that the primary GCT cultures secrete both IL-1beta and MMP-9. The addition of monoclonal antibody (mAb) against IL-1beta partially abrogated, but did not abolish, MMP-9 expression. Our results on gelatin zymography, reverse transcriptase-polymerase chain reaction (RT-PCR), and immunofluorescence showed that GCT stromal cells did not express MMP-9, although treatment with IL-1beta induced MMP-9 expression in a dose-dependent manner, and the secretion peaked 24 h after stimulation and then plateaued. Studies with cycloheximide, a protein synthesis inhibitor, demonstrated that de novo protein synthesis is required for IL-1beta induced MMP-9 expression. Moreover, nuclear run-on analysis has revealed that IL-1beta significantly increased MMP-9 gene transcription in GCT stromal cells. The data suggest that IL-1beta secreted by the multinucleated giant cells in GCT may be one of the factors responsible for the induction of MMP-9 at the transcriptional level in GCT stromal cells in vivo. We conclude that GCT has a self-stimulatory system for the production of MMP-9, and the ability of stromal cells to produce MMP-9 with appropriate stimuli, such as IL-1beta, and possibly in concert with other cytokines may contribute to the aggressive and potentially malignant behavior of GCT.

Is a duplication of 14q32 a new recurrent chromosomal alteration in B-cell non-Hodgkin lymphoma?

Identification of clonal chromosomal abnormalities involving 14q32 and its association with specific histological subtypes of non-Hodgkin lymphoma (NHL) has provided substantial insight to the genetic events leading to the disease. However, in some cases with inferior morphology of tumor cell chromosomes, the additional segment on chromosome 14 remains unidentified by cytogenetic banding techniques alone. To elucidate the origin of the additional chromosomal segment and to correlate the newly determined alterations with histology, metaphases from 15 NHL patients with add(14)(q32) were examined using fluorescence in situ hybridization (FISH) techniques after cytogenetic analysis had been performed. We found the duplication of 14q involving the q32 region in 6 cases with a dup(14) (q32) in 4 cases and a dup(14)(q24q32) in 2 cases. In 8 cases, FISH unveiled known NHL associated translocations; a t(14;18)(q32;q21) in 4 cases, a t(11;14)(q13;q32) in 2 cases, a t(8;14)(q24;q32) and a t(9;14)(p13;q32) in 1 case each. We also noted a t(14;17)(q32;q21) in 1 case. The use of FISH was a valuable asset in determining the origin of the additional material on chromosome 14q32, and helped resolve a group of B-cell NHLs with involvement of a duplicated 14q32 region.

Rearrangements of chromosome band 1p36 in non-Hodgkin's lymphoma.

We studied 850 consecutive cases of histologically ascertained pretreatment non-Hodgkin's lymphoma with cytogenetically abnormal clones. The diagnostic karyotypes revealed that 12% of these cases exhibited structural rearrangements involving chromosome band 1p36. Here, we describe the karyotypes of 53 cases containing a 1p36 rearrangement [often involving translocations of unknown material and presented as add(1)(p36)]. We used fluorescence in situ hybridization to determine the origin of the translocation partners. We report three different recurrent translocations involving 1p36. These include der(1)t(1;1)(p36;q21) (three cases), der(1)t(1;1)(p36;q25) (three cases), and der(1)t(1;9)(p36;q13) (four cases). Using cytogenetic and fluorescence in situ hybridization analyses, we have resolved the translocation partners in 31 cases. Rearrangements of band 1p36 were found among different histopathological subtypes. Alterations of 1p36 never occurred as a sole abnormality, and in 42 of 53 cases, alterations of the band 14q32 were observed. The t(14;18)(q32;q21) translocation was present in 35 cases. The significantly high occurrence of 1p36 breakpoint in structural rearrangements and its involvement in recurrent translocations suggest that the region is bearing gene(s) that are important in lymphomagenesis. Our study also showed that cytogenetically evident deletions were frequent in chromosome 1p, almost always involving the p36 region, whereas duplications were rare and never encompassed the p36 region. Chromosome band 1p36 harbors many candidate tumor suppressor genes, and we propose that one or more of these genes might be deleted or functionally disrupted as a molecular consequence of the rearrangements, thus contributing to lymphomagenesis.

Inactivating mutations and overexpression of BCL10, a caspase recruitment domain-containing gene, in MALT lymphoma with t(1;14)(p22;q32).

Mucosa-associated lymphoid tissue (MALT) lymphomas most frequently involve the gastrointestinal tract and are the most common subset of extranodal non-Hodgkin lymphoma (NHL). Here we describe overexpression of BCL10, a novel apoptotic signalling gene that encodes an amino-terminal caspase recruitment domain (CARD), in MALT lymphomas due to the recurrent t(1;14)(p22;q32). BCL10 cDNAs from t(1;14)-positive MALT tumours contained a variety of mutations, most resulting in truncations either in or carboxy terminal to the CARD. Wild-type BCL10 activated NF-kappaB but induced apoptosis of MCF7 and 293 cells. CARD-truncation mutants were unable to induce cell death or activate NF-kappaB, whereas mutants with C-terminal truncations retained NF-kappaB activation but did not induce apoptosis. Mutant BCL10 overexpression might have a twofold lymphomagenic effect: loss of BCL10 pro-apoptosis may confer a survival advantage to MALT B-cells, and constitutive NF-kappaB activation may provide both anti-apoptotic and proliferative signals mediated via its transcriptional targets.

Deletion of cell division cycle 2-like 1 gene locus on 1p36 in non-Hodgkin lymphoma.

Our laboratories have documented a significantly high occurrence of chromosome 1p36 rearrangements in non-Hodgkin lymphoma (NHL). The cell division cycle 2-like 1(CDC2L1) (also known as TP58 or PITSLRE) gene, a protein kinase implicated in apoptotic signaling, is located at the very distal region of chromosome 1p36 and is likely to be disrupted by structural rearrangements involving 1p36. To determine the molecular consequences of the recurrent involvement of the 1p36 region, we examined metaphases containing 1p36 abnormalities from 31 specimens derived from 26 patients for the possible deletion of CDC2L1 by fluorescence in situ hybridization (FISH) using the TP58clk-1 DNA probe. Twenty-three cases exhibited the loss of CDC2L1 from the abnormal chromosome 1. In 2 of 26 cases, the gene locus was translocated to the partner chromosome, and in four specimens, all derived from one case, CDC2L1 was not deleted. This pilot investigation suggests that 1p36 rearrangements, and consequently the loss of the CDC2L1 gene locus, is important in NHL. This work also opens avenues for further molecular studies and prognostic correlations.

Transcriptional regulation of MMP-9 expression in stromal cells of human giant cell tumor of bone by tumor necrosis factor-alpha.

We determined whether certain factor(s) secreted by multinucleated giant cells, which is of monocyte/macrophage lineage in giant cell tumor of bone (GCT), regulate the induction of matrix metalloproteinase (MMP)-9 expression in mononucleated stromal cells. Our data derived using enzyme linked immunosorbant assays (ELISAs) suggest that the GCT cells in primary culture produce both MMP-9 and tumor necrosis factor-alpha (TNF-alpha). Further, the MMP-9 expression in GCT primary cultures was partially abrogated by neutralizing antibody to TNF-alpha, suggesting that TNF-alpha secretion by the multinucleated giant cells may be one of the factors responsible for the production of MMP-9 by the stromal cells in vivo. In order to confirm this we examined the role of TNF-alpha on the induction of MMP-9 expression in bone GCT stromal cells. These cells express MMP-2, but not MMP-9. However, treatment of these cells with TNF-alpha induced the expression of MMP-9 in a concentration-dependent manner. Kinetic experiments revealed that the secretion of MMP-9 peaked 12 h post TNF-alpha stimulation. Immunofluorescence studies confirmed the expression of MMP-9 after stimulation of GCT stromal cells with TNF-alpha. Further, TNF-alpha-induced MMP-9 expression was completely blocked with neutralizing antibody to TNF-alpha, thereby demonstrating the specificity. In addition, the induction of MMP-9 expression by TNF-alpha was completely abrogated in the presence of cycloheximide, a protein synthesis inhibitor, suggesting that de novo protein synthesis may be required. Nuclear run-on analysis demonstrated that treatment of GCT stromal cells significantly enhanced the MMP-9 gene transcription. Together, our data suggest that TNF-alpha secreted by the multinucleated giant cells up-regulates MMP-9 expression in GCT stromal cells by the induction of certain transcription factors, which in turn enhanced the rate of transcription of MMP-9 gene. These studies also suggest the existence of an essential cell-cell interaction in the regulation of MMP-9 expression in GCT.