Genotype and phenotype in 12 additional individuals with SATB2-associated syndrome.

SATB2-associated syndrome (SAS) is a multisystemic disorder caused by alterations of the SATB2 gene. We describe the phenotype and genotype of 12 individuals with 10 unique (de novo in 11 of 11 tested) pathogenic variants (1 splice site, 5 frameshift, 3 nonsense, and 2 missense) in SATB2 and review all cases reported in the published literature caused by point alterations thus far. In the cohort here described, developmental delay (DD) with severe speech compromise, facial dysmorphism, and dental anomalies were present in all cases. We also present the third case of tibial bowing in an individual who, just as in the previous 2 individuals in the literature, also had a truncating pathogenic variant of SATB2. We explore early genotype-phenotype correlations and reaffirm the main clinical features of this recognizable syndrome: universal DD with severe speech impediment, mild facial dysmorphism, and high frequency of craniofacial anomalies, behavioral issues, and brain neuroradiographic changes. As the recently proposed surveillance guidelines for individuals with SAS are adopted by providers, further delineation of the frequency and impact of other phenotypic traits will become available. Similarly, as new cases of SAS are identified, further exploration of genotype-phenotype correlations will be possible.

Association of the missense variant p.Arg203Trp in PACS1 as a cause of intellectual disability and seizures.

Graphical abstract key: ADHD, attention deficit hyperactivity disorder; ASD, atrial septal defect; DD, developmental delay; EEG, electroencephalogram; Ht, height; ID, intellectual disability; OCD, obsessive-compulsive disorder; OFC, open fontanelle; PDA, patent ductus arteriosis; PFO, patent foramen ovale; VSD, ventricular septal defect; Wt, weight.

Autosomal recessive hereditary spastic paraplegia-clinical and genetic characteristics of a well-defined cohort.

We describe the clinical and genetic features of a well-characterized cohort of patients with autosomal recessive hereditary spastic paraplegia (ARHSP) in the province of Ontario. Patients with documented corticospinal tract abnormalities were screened by whole gene sequencing and multiplex ligation probe amplification for mutations in nine genes known to cause ARHSP. Of a cohort of 39 patients, a genetic diagnosis was established in 17 (44 %) and heterozygous mutations were detected in 8 (21 %). Mutations were most frequent in SPG7 (12 patients), followed by SPG11 (10 patients), PNPLA6 (SPG39, 2 patients), and ZFYVE26 (SPG15, 2 patients). Although there are associations between some clinical manifestations of ARHSP and specific genes, many patients are tested at an early stage of the disease when phenotype/genotype correlations are not obvious. Accurate molecular characterization of well-phenotyped cohorts of patients will be essential to establishing the natural history of these rare degenerative disorders to enable future clinical trials.

Familial Simpson-Golabi-Behmel syndrome: studies of X-chromosome inactivation and clinical phenotypes in two female individuals with GPC3 mutations.

Simpson-Golabi-Behmel syndrome (SGBS) is an overgrowth/multiple congenital anomalies syndrome with an X-linked inheritance. Most cases of SGBS are attributed to mutations in the glypican 3-gene (GPC3), which is highly expressed in the mesodermal embryonic tissues and involves in a local growth regulation. Typical clinical features include pre/postnatal overgrowth, developmental delay, macrocephaly, characteristic facies with prominent eyes and macroglossia, diaphragmatic hernia, congenital heart defects, kidney anomalies, and skeletal anomalies. Obligate carrier females with GPC3 mutations are usually asymptomatic or with mild symptoms. It is thought that skewed X-inactivation is the underlining mechanism for the female patients to present with findings of SGBS. We identified three siblings with typical SGBS (two male and one female cases) and their mother with very mild symptoms in a family carrying c.256C>T (p.Arg86X) mutation in GPC3. X-inactivation studies on the androgen-receptor gene (AR) and the Fragile XE (FRAXE) gene were performed with blood, buccal swabs, and fibroblasts in the carrier females. The studies with blood showed moderately skewed X-inactivation with paternal X-chromosome being preferentially inactivated (71-80% inactivated) in the female patient with SGBS and no skewing was shown in the mother with very mild symptoms. The X-inactivation studies in the mother showed inactivation of the X-chromosome with the mutation by 57%. This suggests that loss of the functional GPC3 protein by 43% is closed to the threshold to develop the SGBS phenotype. Studies with buccal swabs and fibroblasts failed to show different X-inactivation patterns between the two female individuals.

The clinical and genetic epidemiology of neuronal ceroid lipofuscinosis in Newfoundland.

The neuronal ceroid lipofuscinoses (NCLs) are the commonest neurodegenerative disorders of children. The aims of this study were to determine the incidence of NCL in Newfoundland, identify the causative genes, and analyze the relationship between phenotype and genotype. Patients with NCL diagnosed between 1960 and 2005 were ascertained through the provincial genetics and pediatric neurology clinics. Fifty-two patients from 34 families were identified. DNA was obtained from 28/34 (82%) families; 18 families had mutations in the CLN2 gene, comprising five different mutations of which two were novel. One family had a CLN3 mutation, another had a novel mutation in CLN5, and five families shared the same mutation in CLN6. One family was misdiagnosed, and in two, molecular testing was inconclusive. Disease from CLN2 mutations had an earlier presentation (p = 0.003) and seizure onset (p < 0.001) compared with CLN6 mutation. There was a slower clinical course for those with CLN5 mutation compared with CLN2 mutation. NCL in Newfoundland has a high incidence, 1 in 7353 live births, and shows extensive genetic heterogeneity. The incidence of late infantile NCL, 9.0 per 100,000 (or 1 in 11,161) live births, is the highest reported in the world.

Structural basis for autoinhibition of the Ephb2 receptor tyrosine kinase by the unphosphorylated juxtamembrane region.

The Eph receptor tyrosine kinase family is regulated by autophosphorylation within the juxtamembrane region and the kinase activation segment. We have solved the X-ray crystal structure to 1.9 A resolution of an autoinhibited, unphosphorylated form of EphB2 comprised of the juxtamembrane region and the kinase domain. The structure, supported by mutagenesis data, reveals that the juxtamembrane segment adopts a helical conformation that distorts the small lobe of the kinase domain, and blocks the activation segment from attaining an activated conformation. Phosphorylation of conserved juxtamembrane tyrosines would relieve this autoinhibition by disturbing the association of the juxtamembrane segment with the kinase domain, while liberating phosphotyrosine sites for binding SH2 domains of target proteins. We propose that the autoinhibitory mechanism employed by EphB2 is a more general device through which receptor tyrosine kinases are controlled.

Tubular carcinoma of the breast: sensitivity of diagnostic techniques and correlation with histopathology.

OBJECTIVE: Our objective was to assess our experience in diagnosing pure tubular carcinoma of the breast and to correlate the radiologic and histopathologic features. MATERIALS AND METHODS: A retrospective review of 932 consecutive cases of proven breast cancer diagnosed between 1990 and 1997 revealed 78 cases (8.4%) of tubular carcinoma in 69 patients. Clinical, imaging, cytologic, and histologic findings were analyzed. RESULTS: Mammography revealed tubular carcinoma in 68 (87%) of the 78 cases. Sonography showed tubular carcinoma in all 38 cases in which it was used; nine of these lesions were mammographically occult. These nine lesions were slightly, but not significantly (p < .05), smaller than the 29 lesions that had also been detected on mammography. Large core needle biopsy was performed in 22 patients (sensitivity, 91%). At biopsy, diagnoses were malignant (n = 16 [73%]), suspicious (n = 4 [18%]), atypia (n = 1 [4.5%]), and benign (n = 1 [4.5%]). Fine-needle aspiration biopsy was used to evaluate 36 cases of tubular carcinoma (sensitivity, 50%); cytologic diagnoses were malignant (n = 15 [42%]), suspicious (n = 3 [8%]), atypia (n = 10 [28%]), and benign (n = 8 [22%]). Only 15 (19%) of the 78 tubular carcinomas were palpable. Other tumors were detected within the excised tissue in 47 of the patients (68%); of these other types of lesions, ductal carcinoma in situ was found most often. CONCLUSION: Most cases of tubular carcinoma can be revealed by mammography; for mammographically occult tubular carcinoma, sonography can be performed. The rate of accuracy for determining the presence of tubular carcinoma is higher with large core needle biopsy than with fine-needle aspiration biopsy. Finally, when tubular carcinoma is diagnosed, other histologic types of carcinoma often occur in the same breast.

Characterization of the CDR3 region of rearranged alpha heavy chain genes in human fetal liver.

The human fetal liver is an early site for B cell development. Pre B cells are first detectable in human fetal life at 8 weeks of gestation, when the rearrangement of the mu heavy chain genes starts. In this study we characterize the CDR3 region of rearranged alpha heavy chain transcripts from four human fetal livers ranging from 8 to 11 weeks of gestation. Each fetal liver showed a limited number of variations in CDR3 sequences compared with adult peripheral blood mononuclear cells (PBMC). Sequence analysis of 91 clones demonstrated that there was no preference for the usage of a certain JH gene segment, whereas a preference for usage of DH family genes, DXP and DLR, was seen in most cases during early fetal life. This is the first study where rearranged alpha heavy chain genes in fetal liver have been characterized. Our data suggest that the usage of JH genes is random, while there is a preference for DH family genes in human fetal liver.

Hepatocyte growth factor/scatter factor stimulates chemotaxis and growth of malignant mesothelioma cells through c-met receptor.

Hepatocyte growth factor (HGF) and its receptor c-met are present in several human tissues but their expression in mesothelial cells has not been examined. In this study, we have investigated the expression of HGF and c-met in normal human mesothelial cells and 11 human malignant mesothelioma cell lines. Using RT-PCR and Western blotting we found that HGF is produced by 3/11 mesothelioma cell lines whereas c-met is expressed in 11/11 mesothelioma cell lines. In addition, c-met expression was also found in 6/6 cell samples obtained from pleural fluids of patients with mesothelioma. In contrast, neither normal cultured mesothelial cells nor mesothelial cells obtained directly from patients without mesothelioma expressed HGF nor c-met. We have also analysed the biological function of HGF and c-met in mesothelioma cell lines. Recombinant human (rh) HGF stimulated both directional (chemotactic) and random (chemokinetic) motility in all mesothelioma cell lines tested. Furthermore, mesothelioma serum free conditioned medium containing HGF stimulated mesothelioma cell migration. This effect could be blocked in the presence of neutralizing anti-HGF monoclonal antibodies (MAbs) in the assay. Addition of HGF to mesothelioma cells cultured on collagen type IV was associated with induction of bipolar shape and protrusion of prominent pseudopodia. We have also found that rhHGF was mitogenic for mesothelioma cells. Our findings suggest that expression of HGF/c-met is involved not only in mesothelioma progression but also in its growth and migration and that c-met expression found in mesothelioma cells taken directly from patients may be of diagnostic importance.

Platelet-derived growth factor (PDGF) BB acts as a chemoattractant for human malignant mesothelioma cells via PDGF receptor beta-integrin alpha3beta1 interaction.

Platelet-derived growth factor BB (PDGF BB) and the PDGF receptor beta are expressed on mesothelioma cells, but their biological function has not yet been defined. In the present study we used Boyden chambers fitted with filters coated with the adhesive matrix proteins fibronectin, laminin, collagen type IV or the nonmatrix adhesive molecule poly-L-lysine (PLL). Mesothelioma cells migrated towards PDGF BB at concentrations ranging from 0.78 to 12.5 ng/ml if matrix proteins were present as adhesive substrates. This migration was integrin dependent since the same cells failed to migrate if the adhesive interactions necessary for migration were provided by molecules other than integrins. Migration of mesothelioma cells on fibronectin, laminin or collagen-type IV in response to PDGF BB was inhibited if the cells were pretreated with blocking antibodies to alpha3beta1 integrin. These findings describe for the first time PDGF BB as a chemoattractant for malignant mesothelioma cells and that collaboration between PDGF receptor beta and integrin alpha3beta1 is necessary for the motile response of these cells to PDGF BB.

Direct and sequential switching from mu to epsilon in patients with Schistosoma mansoni infection and atopic dermatitis.

Immunoglobulin isotype switching to IgE in patients infected with Schistosoma mansoni and patients with atopic dermatitis was studied. Patients with parasitic infections or allergic diseases have a higher production of IgE. We found a four-fold increased production of I epsilon RNA in both patient groups as compared to control donors. The increased expression of germ-line transcripts correlates with higher serum IgE levels. Nested primer polymerase chain reaction was used to generate S mu/S epsilon fragments from DNA of patient peripheral blood mononuclear cells. Twenty-nine out of fourty sequenced switch fragments had undergone direct joining from S mu to S epsilon whereas seven fragments showed mono sequential switching from S mu via either S mu, S gamma2, S gamma4 or S epsilon to S epsilon and four fragments demonstrated double sequential switch: S mu/S mu/S gamma1/S epsilon, S mu/S gamma2/S epsilon/S epsilon or S mu/S gamma1/ S gamma2/S epsilon. The sequential switching had occurred either via deletions or inversions. Mapping of the breakpoints showed hot spots for recombination within S mu, S gamma1 and S epsilon. To our knowledge, this is the first in vivo study in humans demonstrating that switching to IgE can occur from sequential rearrangements via gamma1, gamma2 or gamma4.

Studies of the molecular basis of IgA production, subclass regulation and class-switch recombination in IgA nephropathy patients.

IgA nephropathy (IgAN), the most common form of glomerulonephritis, is characterized by normal to elevated levels of serum IgA. In order to understand the molecular mechanism(s) involved in the production of IgA in IgAN, peripheral blood mononuclear cells (PBMC) from these patients were analysed in this study. IL-10, transforming growth factor-beta 1 (TGF-beta 1) and CD40 have previously been shown to be involved in IgA production. We show here that CD40L expression was increased three-fold in these patients. However, expression of TGF-beta 1 in serum levels was comparable to controls. In vitro stimulation of PBMC with a polyclonal activator resulted in a three-fold increase in synthesis of both IgA subclasses, with a preference for IgA1 RNA. In situ hybridization studies also showed a three-fold increase in the numbers of IgA1- and IgA2-producing cells, but the subclass distribution was similar to the controls. Furthermore, using the nested primer polymerase chain reaction (PCR) for amplifying switch (S mu/S alpha) breakpoints we could demonstrate that in unstimulated PBMC the switch frequency did not differ from that of control donors. Sequence analysis of the amplified switch breakpoints and the I alpha regulatory region from patients showed no structural abnormality. Although we have previously demonstrated a correlation to in vivo germ-line RNA expression and class switching, no I alpha transcripts were detected in unstimulated PBMC from these patients. However, stimulation of PBMC with TGF-beta 1 resulted in I alpha production. Taken together, results from in vivo and in vitro studies suggest that increased cytokine production and hyperresponsiveness to polyclonal stimulation may play an important role in the increased synthesis of IgA. The preference for IgA1 is due to increased production of IgA1 per cell, and the absence of I alpha RNA indicates that additional defect(s) in immune regulation may play an important role in the pathogenesis of IgAN.

Genetics of IgA deficiency.

IgA deficiency is the most common humoral defect in man and results in an increased susceptibility to respiratory tract and gastrointestinal infections. Both clinical and genetic data support a close relationship with common variable immunodeficiency, a disease which involves not only IgA and IgG production, but also, in half of the patients, IgM. It is likely that the two disorders represent an allelic condition with a variable expression of a common gene defect which is thought to be involved in the regulation of immunoglobulin class switching. It is possible that a single, autosomally inherited gene with a limited penetrance is responsible for the development of both these defects.

Molecular analysis of IgA deficiency. Evidence for impaired switching to IgA.

The most common form of primary immunodeficiency is IgA deficiency (IgAD). However, the molecular basis of this disease remains elusive. Therefore, to address this issue we made a systematic analysis of the molecular events leading to IgA production. B lymphocytes that produce IgA have undergone somatic rearrangement that joins the switch (S) mu to S alpha region with deletion of the intervening sequences. Examination of the resulting S mu/S alpha junctions in unstimulated PBMC from IgAD patients by nested primer PCR revealed a significant decrease in the number of the S mu/S alpha fragments. To obtain the antisense primers to generate the S mu/S alpha fragments, we sequenced the human S alpha 1 and the downstream region extending to the C alpha 1 locus. Similar to previously reported switch sequences, we also found the S alpha 1 to be predominantly composed of pentameric repeats GAGCT and GGGCT. The decrease in the number of S mu/S alpha fragments is consistent with a profound decrease in the C alpha membrane mRNA expression in unstimulated PBMC, as well as in the C alpha mRNA levels and IgA production in PWM-stimulated PBMC. Sequence analysis of the switch junctions from IgA-producing cell lines, control donors, and an IgAD patient showed direct joining in 8 of 9 cases examined. TGF-beta 1, previously shown to be the switch factor for human and mouse IgA, was also examined. No difference in the TGF-beta 1 mRNA levels in unstimulated PBMC between the control subjects and the IgAD patients were detected. Our findings indicate that the failure to switch to IgA-producing B lymphocytes, or an impaired survival of such cells, may be an important molecular mechanism in IgAD.

Expression of Bruton's agammaglobulinemia tyrosine kinase gene, BTK, is selectively down-regulated in T lymphocytes and plasma cells.

The gene mutated in the human disease, X-linked agammaglobulinemia (XLA), is related to the Src gene family of cytoplasmic protein-tyrosine kinases and is designated Btk (Bruton's agammaglobulinemia tyrosine kinase; formerly Atk/Bpk; the human gene is denoted BTK, using capital letters according to the kinase nomenclature). We have recently reported that this gene is expressed in B lymphocytes and that the specific mRNA was undetectable in T cells using Northern blotting. Further analyses of different sources of B and T lymphocytes confirmed this pattern. However, BTK transcripts were undetectable in four plasmacytoma lines. Moreover, as virtually normal amounts of BTK transcripts were found in PBMC from two patients carrying a point mutation in BTK, despite low B cell numbers, we anticipated that the gene would also be expressed in cells of other lineages. The erythroleukemia cell line K-562, the promyelocytic line HL-60 and the histiocytic lymphoma line U-937 were found to have BTK mRNA levels comparable to B cells. BTK mRNA was also detected in monocytes from healthy donors as well as in the human immature basophilic cell line KU812, in the human mast cell leukemia cell line HMC-1 and in the CD34 expressing myeloblast KG-1. A similar expression pattern was obtained when BTK protein was analyzed by immunoprecipitation and Western blotting. Using a polymerase chain reaction-based analysis, a small amount (less than 1% of the level in B cells) of BTK mRNA was identified in T lymphocytes. Our findings are compatible with a general expression of the BTK gene in hematopoietic cells, except in T lymphocytes and plasma cells, in which the transcript level is selectively down-regulated.

In vivo expression of human immunoglobulin germ-line mRNA in normal and in immunodeficient individuals.

Previous in vitro studies suggest that transcription of the unrearranged immunoglobulin switch region and its 5' flanking region precedes isotype switching. These transcripts, which are devoid of a variable region, contain unique exons and are called germ-line (GL) mRNA. A crucial point in this regard is whether such transcripts could be detected in vivo, and if their expression correlates with immunoglobulin class switching in health and disease. To understand the in vivo role of this transcriptional activity we have adapted the reverse transcription-polymerase chain reaction (PCR) to analyse the GL transcripts from unstimulated peripheral blood mononuclear cells (PBMC) in healthy individuals and in different immunological diseases. Furthermore, mononuclear cells from different human organs were also analysed. We report here that GL (I alpha, I gamma and I epsilon used to designate the GL mRNA for IgA, IgG and IgE, respectively) mRNA are expressed differentially during ontogeny of B cells. Unexpectedly, no difference of I alpha mRNA expression between the PBMC and the secondary lymphoid organs was detected. Rather, the levels of GL transcripts were correlated to the number of sIgM+ cells. GL mRNA of all three isotypes could be detected in PBMC from healthy donors, whereas there was a decrease of specific GL transcript synthesis in individuals with immunoglobulin deficiency. Furthermore, during the in vivo immune response in a parasitic infection, we could demonstrate an induction of GL I epsilon mRNA during in vivo immune response. Concomitantly, there was also increased synthesis of productive epsilon transcripts. These findings implicate a potential role of GL transcription during in vivo immunoglobulin class switching.

Human immunoglobulin germline alpha(IgA) transcripts are expressed in vivo and their level correlates to class switching.

Immunoglobulin isotype switching is preceded by transcription of exons located 5' of the immunoglobulin switch regions. These exons are referred to as 'I-exons' and transcription of these regions is believed to be an essential step in switch recombination. Such mRNA species lack a variable portion and are denoted 'germline' transcripts. We have previously identified I-regions in man and we have now investigated the in vivo expression of human germline transcripts. Germline alpha transcripts (containing an I alpha exon) were expressed in various lymphoid organs, including peripheral blood mononuclear cells. Decreased levels of germline transcripts were found in immunoglobulin deficiency diseases. These findings are compatible with an in vivo role of germline transcription in isotype switching and may contribute to the understanding of mechanisms underlying deficiency diseases.