Deletion of MAP2K2/MEK2: a novel mechanism for a RASopathy?

RASopathies are a class of genetic syndromes caused by germline mutations in genes encoding Ras/mitogen-activated protein kinase (Ras/MAPK) pathway components. Cardio-facio-cutaneous (CFC) syndrome is a RASopathy characterized by distinctive craniofacial features, skin and hair abnormalities, and congenital heart defects caused by activating mutations of BRAF, MEK1, MEK2, and KRAS. We define the phenotype of seven patients with de novo deletions of chromosome 19p13.3 including MEK2; they present with a distinct phenotype but have overlapping features with CFC syndrome. Phenotypic features of all seven patients include tall forehead, thick nasal tip, underdeveloped cheekbones, long midface, sinuous upper vermilion border, tall chin, angular jaw, and facial asymmetry. Patients also have developmental delay, hypotonia, heart abnormalities, failure to thrive, obstructive sleep apnea, gastroesophageal reflux and integument abnormalities. Analysis of epidermal growth factor-stimulated fibroblasts revealed that P-MEK1/2 was ∼50% less abundant in cells carrying the MEK2 deletion compared to the control. Significant differences in total MEK2 and Sprouty1 abundance were also observed. Our cohort of seven individuals with MEK2 deletions has overlapping features associated with RASopathies. This is the first report suggesting that, in addition to activating mutations, MEK2 haploinsufficiency can lead to dysregulation of the MAPK pathway.

Gonosomal mosaicism for an NF1 deletion in a sperm donor: evidence of the need for coordinated, long-term communication of health information among relevant parties.

BACKGROUND: Screening of gamete donors can reduce but cannot eliminate the risks for medical problems in donor-conceived offspring. We present a case of gonosomal mosaicism discovered in an anonymous sperm donor after receiving two reports of neurofibromatosis type 1 (NF1) in donor-conceived offspring, to illustrate that long-term, systematic investigation of health issues in donors and offspring can be invaluable to the welfare of these individuals. METHODS: A repeat physical evaluation and ophthalmology examination were performed on the donor. DNA samples were examined by RTPCR fragment analysis, multiplex ligation-dependent probe amplification (MLPA) and targeted array-comparative genomic hybridization (aCGH). RESULTS: Gonosomal mosaicism for a deletion mutation in the NF1 gene was identified in 20% of sperm and a smaller percentage of lymphocytes. CONCLUSIONS: Long-term communication of medical information among donors, recipients and donor-conceived offspring is beneficial for the health management of all parties. Development of a secure, coordinated data system is critical to achieving this goal. Recommendations are provided for management and communication of critical information based on this experience.

Nonspecific complement activation by streptococcal structures. I. Re-evaluation of HLA cytotoxicity inhibition.

A number of experiments have suggested that there is an antigenic relationship between the HLA complex and streptococcal bacterial structures. Using inhibition of cytotoxicity of HLA antisera as our assay system, it was demonstrated that the inhibitory effect on HLA cytotoxicity by streptococcal antigens is, in reality, due to activation and consumption of components of the alternate complement pathway. In addition, antisera prepared against streptococcal membrane antigens had no cytotoxic effect on a large panel of human lymphocytes, nor did these antisera exhibit immunofluorescent staining of lymphocytes directly. These experiments are compatible with our concept that the HLA complex may have evolved through selective evolutionary pressure as a means of escaping bacterial mimicry.

Lymphocytic responses to streptococcal antigens in glomerulonephritic patients.

The lymphocytes from patients with progressive glomerulonephritis showed significant inhibition of cell migration in the presence of group A streptococcal particulate antigens. Marked increases in the level of DNA synthesis of these lymphocytes were also observed after contact with these antigens. Lymphocytes from patients with unrelated renal disorders exhibited minimum reactivity to streptococcal antigens.

Cellular transplantation in the treatment of experimental hepatic failure.

The survival of Lewis rats with D-galactosamine-induced fulminant hepatic failure was prolonged if they were given intraperitoneal injections of single-cell suspensions of liver or bone marrow cells from normal rats. Suspensions of liver cells were also effective in prolonging the survival of rats with ischemia-induced hepatic necrosis. The liver cells did not act by repopulating the recipient liver.

Sensitization to low dose 5-fluorouracil. Subsequent enhancement of its systemic antitumor effect in the rat.

This report describes a novel method of immunochemotherapy; the active immunization to the drug 5-fluorouracil (5-FU) with enhanced antitumor activity resulting from its subsequent systemic administration. Two metastasizing carcinomas in the Fischer strain (F344) rat have been used: a chemically induced bladder carcinoma (FBCa) and a spontaneous mammary adenocarcinoma (MACa). Both tumors grow rapidly and result in 100% mortality within 10 wk of implantation. Neither tumor is sensitive to systemic 5-FU alone. Intradermal sensitization to 5-FU before FBCa tumor implantation, followed by 5-FU administered systemically, resulted in significant tumor regression and improvement in survival with eradication of all tumor and cure in 20% of animals. A similar antitumor effect was observed with the MACa. A comparable drug effect was observed when methotrexate sensitization was given before FBCa implantation followed by systemic MTX. Specificity to the sensitizing drug was demonstrated by the lack of effect of sensitization with either 5-FU or MTX unless followed by systemic therapy with the requisite sensitizing agent. Sensitization to 5-FU has also been assessed after FBCa implantation followed by resection of the local tumor. Resection was performed after distant tumor metastases had occurred, and was followed by systemic 5-FU therapy. Whereas tumor resection alone failed to cure any animal, sensitization to 5-FU increased cure rate fourfold over animals receiving systemic 5-FU alone. Antibody to 5-FU in the sera of sensitized animals has been suggested by an immunoenzymatic staining technique and its specificity confirmed in a radioimmunoassay. It is postulated that a combination of the systemic agent and the antibody elicited to it by sensitization produces the significant antitumor effect observed. The antitumor effect observed with this new approach to immunochemotherapy warrants further experimental and clinical study.

16, 16 Dimethyl prostaglandin E2 prevents the development of fulminant hepatitis and blocks the induction of monocyte/macrophage procoagulant activity after murine hepatitis virus strain 3 infection.

16, 16 Dimethyl prostaglandin E2 (dmPGE2), a known cytoprotective agent, was examined for its ability to alter the course of fulminant hepatitis in an experimental model of fulminant viral hepatitis, murine hepatitis murine hepatitis type 3 (MHV-3). Fully susceptible BALB/cJ mice, infected with 100 50% lethal doses (LD50) of MHV-3 developed histologic and biochemical evidence of fulminant hepatitis, as evidenced by massive hepatic necrosis with hypoglycemia, metabolic acidosis, and a markedly elevated serum alanine aminotransferase (ALT) (mean, 1,402 +/- 619 IU/liter). In contrast, animals treated with dmPGE2 either before or after infection (up to 48 h) demonstrated a marked reduction in both histologic and biochemical evidence of liver damage as characterized by normal blood glucose, total CO2, and ALT determinations (mean ALT, 63 +/- 40 IU/liter). Treatment of infected mice with PGF2 alpha demonstrated no cytoprotective effects. High titers of infectious virus were recovered from the livers of both dmPGE2-treated and -untreated animals throughout the course of infection. In a parallel in vitro study, dmPGE2 (10(-4)-10(-8) M) demonstrated a similar cytoprotective effect on monolayers of isolated cultured hepatocytes from fully susceptible BALB/cJ mice infected at a multiplicity of infection of 0.1, 1.0, and 10.0. In addition, splenic macrophages recovered from infected and untreated BALB/cJ mice demonstrated a marked augmentation in procoagulant activity (PCA) from a basal 10 +/- 5 mU/10(6) splenic macrophages to a maximum of 615 +/- 102 mU/10(6) splenic macrophages, whereas no increase in macrophage PCA was detected in infected animals treated with dmPGE2. These results suggest that dmPGE2, without detectably altering viral replication or infectivity in vivo, confers a marked cytoprotective effect on hepatocytes both in vivo and in vitro, and prevents the induction of macrophage PCA in vivo in fully susceptible BALB/cJ mice after murine hepatitis virus type 3 infection.

Cellular reactivity studies to streptococcal antigens. Migration inhibition studies in patients with streptococcal infections and rheumatic fever.

The question of whether hypersensitivity to streptococcal antigens plays a role in the pathogenesis of the nonsuppurative sequelae of streptococcal infections remains at present unclear. As a first step in the approach to this question, the degree of cellular reactivity of peripheral blood leucocytes to streptococcal antigens was investigated in a number of rheumatic fever patients, patients with uncomplicated streptococcal infections, as well as normal healthy subjects. Using the in vitro technique for the inhibition of capillary migration of peripheral blood leucocytes as an index of the degree of sensitivity to streptococcal antigens, the results indicate that patients with acute rheumatic fever exhibit an exaggerated cellular reactivity to these antigens and in particular to streptococcal cell membrane antigens. This abnormal response to streptococcal membrane antigens appears to persist in rheumatic subjects for at least 5 yr after the initial attack of rheumatic fever. Only Group A streptococcal membrane antigens elicited this unusual response in rheumatic subjects, since the cellular reactivity to Group C and D streptococcal membranes was the same in all groups. Patients with evidence of valvular disease exhibited the same degree of cellular reactivity to these antigens as did patients without clinical evidence of rheumatic heart disease. The nature of the antigens responsible for the observed cellular response remains unknown. Enzymatic treatment of streptococcal cell walls and membranes designed to remove type-specific M proteins did not alter the observed cellular reactivity to the streptococcal antigens. The finding that an abnormal cellular response to certain streptococcal antigens is present only in rheumatic patients suggests that cell-mediated factors may play an important role in the disease process.

Allogeneic hepatocyte transplantation in the rat spleen under cyclosporine immunosuppression.

An innovative approach for stimulating the rapid growth of allogeneic hepatocytes implanted into splenic tissue with maintenance of the structural integrity is described. Single cell suspensions of hepatocytes from normal male ACI-strain rats (RTIa) were injected (2 X 10(6) cells) into the spleen of allogeneic male Fischer (RTI1) recipient rats. A 70% partial hepatectomy (PH) was performed at the same time as hepatocyte transplantation. Animals were treated for 4 days prior to, and 1 day after, transplantation with a feeding regimen containing 0.05% 2-acetylaminofluorene (AAF) to inhibit regeneration of the residual host liver. Animals received cyclosporine (CsA) 3 mg/kg/day s.c. posttransplantation. Histological examination of a standard longitudinal section of the recipient spleen two days posttransplant revealed an approximately 0.54-mm2 area replaced by hepatocytes. By 7 days this had increased to 0.97 +/- .15 mm2. Without CsA administration, hepatocytes were undetected at 7 days. Both PH and AAF treatment were necessary for successful colonization and sustained proliferation. Withdrawal of CsA treatment at 10 days after transplantation resulted in rapid rejection of established hepatocytes. This study demonstrates that rapid colonization of the rat spleen with allogeneic hepatocytes can be achieved, and that the viability and structural integrity of these transplanted cells can be maintained for at least 14 days using cyclosporine immunosuppression.

Selective immune stimulation during induction of allograft tolerance in the rat by radical immunosuppression.

Recent investigation of a biologically active synthetic polymer, NED 137, has demonstrated its ability to induce a B cell differentiation response to certain antigens, even in the presence of T cell depletion. In this report, the effect of T cell depletion combined with NED 137, on skin allografting, is explored in the Lewis strain rat. Animals were T cell-depleted by thymectomy, total body irradiation, and syngeneic marrow repopulation. They were then challenged with skin allografts +/- NED 137 treatment. Graft survival was significantly prolonged in the T-depleted rats regardless of treatment with NED 137. The drug did not increase the immune response to donor antigen as measured by in vivo lysis of 51Cr-labeled cells. Immunization with heterologous erythrocytes produced a low level of differentiation of IgM-producing cells in the T cell-depleted skin allografted group, but in contrast the T-depleted NED 137-treated rats had a normal response to immunization. These data suggest that selective stimulation of the humoral component of the immune response is feasible at a time when T cell-mediated function has been radically suppressed, without producing adverse effects on allograft survival.

Allogeneic and xenogeneic hepatocyte transplantation in experimental hepatic failure.

Previous studies have demonstrated the efficacy of syngeneic hepatocyte transplantation in the treatment of D-galactosamine-induced acute hepatic failure in Lewis strain rats. This report describes the efficacy and immunological consequences of allogeneic and xenogeneic hepatocyte transplantation in the same model. The i.p. administration of allogeneic (minor and major histoincompatibility) hepatocytes or xenogeneic (rabbit or porcine) hepatocytes at a dose of 4 x 10(7) cells/rat given at 48 hr after toxin all resulted in significant improvement in survival compared to that of controls, and also comparable to the results obtained with syngeneic hepatocyte transplantation. Sensitization to i.p. allogeneic (WF) hepatocyte administration was demonstrated by in vivo 51Cr release, indirect immunofluorescent technique, and accelerated skin allograft rejection. Similarly, the in vivo 51Cr release assay was able to detect sensitization to porcine hepatocytes. Despite evidence of immunogenicity, redosing with either WF or porcine hepatocytes resulted in no overt toxicity. Furthermore, presensitization by either WF hepatocytes or skin allografts did not adversely affect survival after WF hepatocyte treatment in D-galactosamine-induced hepatic failure in Lewis strain rats. These data demonstrate that histocompatibility is not a constraint to successful hepatocyte transplantation and that repeated treatments are potentially safe and efficacious despite sensitization.

Reversal of cyclosporine-induced mortality with a synthetic polymeric immunostimulant in a murine model of fecal peritonitis.

We have been investigating the effects of a synthetic immunostimulative polymer known as copovithane (Cpv). This agent appears to enhance humoral immunity in untreated and cyclosporine-immunosuppressed mice and is nontoxic in rodents and man. The purpose of this study was to determine whether cyclosporine (CsA) is deleterious to survival in a murine cecal ligation, puncture, and excision (CLPE) model of fecal peritonitis, and--if so--whether this effect could be ameliorated by Cpv without interfering with skin allograft acceptance. Cpv significantly prolongs survival in the CLPE model; the optimal dose for this effect was found to be 100 mg/kg. CsA was found to have a significant and deleterious effect on survival at several dosage levels when administrated 48 and 24 hr before cecal ligation, and immediately before and 16 hr after cecal ligation. Using a dose of CsA sufficient for skin allograft acceptance and the same schedule of administration outlined above, Cpv 100 mg/kg was administered 48 hr prior to cecal ligation. Mice treated with CsA plus Cpv had significantly longer survival than mice treated with CsA alone; furthermore, the survival of CsA-plus-Cpv-treated animals was not significantly different from that of saline-treated controls. Acceptance and survival of H-2 incompatible skin allografts in mice treated with CsA were not affected by Cpv 100 mg/kg/week. We conclude that CsA-induced mortality in the CLPE model can be abrogated by Cpv without adversely affecting skin allograft survival. It may eventually be possible to reduce the incidence of septic complications in clinical allotransplantation by prophylactically administering Cpv to patients on CsA immunosuppression.

Ketonic diet in the management of pyruvate dehydrogenase deficiency.

Two brothers, aged 11 years 6 months and 2 years 3 months, with psychomotor and growth retardation, episodes of weakness, ataxia, ophthalmoplegia, and elevated levels of blood pyruvate were shown to have a deficiency in the pyruvate dehydrogenase complex (PDH). When they ate a diet high enough in fats to cause ketonemia but not acidosis, there was a fall in blood pyruvate levels, a decrease in the frequency and severity of the episodes of neurological deterioration, an increased rate of growth and development in the younger brother, and increased strength and endurance in the older one. The possibility of dietary treatment makes the early diagnosis of PDH deficiency more important. Determination of blood pyruvate and lactate levels following a standard glucose meal (glucose-pyruvate test) appears to be the most reliable screening test for this condition.

A patient with an interstitial deletion of the proximal portion of the long arm of chromosome 4.

A patient with a proximal deletion of the long arm of chromosome 4 is presented. This patient and 6 others previously described appear to have similar findings of moderate to severe developmental delay, small size, small hands and feet, and similar facial appearance. These patients appear to be quite different from those with more distal 4q deletions.

De Barsy syndrome: report of a case, literature review, and elastin gene expression studies of the skin.

Several "progeroid" syndromes have now been identified. The De Barsy syndrome is an autosomal recessive syndrome of dwarfism, mental deficiency, an "aged" appearance at birth, abnormal elastic fibers on skin biopsy, and lax skin, large helices, eye abnormalities, lax joints, hypotonia, and athetoid posturing. We report one case and review 11 cases from the literature. To understand the abnormal appearance of the elastic fibers on biopsy, we performed elastin gene expression studies on fibroblasts cultured from our patient's skin. Molecular hybridization studies revealed reduced elastin mRNA steady-state levels as compared with age matched control individuals. Assuming normal rates of mRNA translation, reduced elastin synthesis would occur. Diminished dermal elastin content could explain the altered cutaneous elasticity, decreased elastic fibers in the skin, and many clinical manifestations of individuals with this condition.

New skeletal dysplasia with unique brachydactyly.

We report on 2 male propositi, their mothers, and a maternal aunt with a new skeletal dysplasia associated with a unique pattern of digital malformation, variable mild short stature, and mild bowleg with proximal overgrowth of the fibula. The digital malformations comprise a pattern of brachydactyly which includes short, abducted thumbs, short index fingers, and markedly short, abducted great toes. The radiographic findings include hypoplastic thumbs and great toes with short first metacarpals and first metatarsals, absent distal phalanges of the index fingers and second toes, and coalescence of the carpal and tarsal bones. Radiographs of the long bones show mild metaphyseal and epiphyseal irregularity, tibial spurs, and relative elongation of the fibulae. The males are very similarly affected whereas the females show phenotypic variation and are generally less severely affected. The family histories from 2 fairly extensive pedigrees suggest X-linked dominant inheritance.

Interstitial deletion of the long arm of chromosome 6 associated with unusual limb anomalies: report of two new patients and review of the literature.

We report on unusual manifestations in 2 unrelated children with interstitial deletion of 6q, with nearly identical breakpoints of 6q16.2q23.1 and 6q16.3q22.3. Major findings include growth retardation, profound developmental delay, microcephaly, facial anomalies, sparse hair, congenital heart defects, and striking hand malformations. Discordant anomalies were duodenal atresia and hypoplastic genitalia in 1 child. Split-hand defect, polydactyly, gastrointestinal anomalies, and ectodermal dysplasia have not been described previously in children with 6q deletion. The presence of hand malformations in 2 children with similar deletion breakpoints strongly suggests that this is a candidate region for one or more genes involved in limb development. Comparison of the clinical findings of other patients with 6q2 deletion suggests a recognizable phenotype.

A 48,XXXX female with absence of ovaries.

A 16 1/2-year-old phenotypic female had primary amenorrhea, mild mental retardation, radioulnar synostosis, and other minor anomalies. Chromosome constitution of leukocytes and skin fibroblasts was 48,XXXX. Plasma levels of gonadotropins were increased, and those of estrogens, decreased consistent with ovarian failure. Laparoscopy showed a small midline uterus, and 2 fallopian tubes, and fimbriae. Neither ovaries nor gonadal streaks were seen on either side. This patient appears to represent the first instance of 48,XXXX aneuploidy with documented absence of ovaries.

Correlation of linkage data with phenotype in eight families with Stickler syndrome.

The clinical findings of eight families with Stickler syndrome were analyzed and compared with the results of linkage studies using a marker for the type II collagen gene (COL2A1). In six families, there was linkage of the phenotype to COL2A1. The manifestations of the affected individuals were similar to those of the original Stickler syndrome family [Stickler et al., Mayo. Clin. Proc. 40:433-455, 1965] and resembled the phenotype of the previously reported individuals or families with Stickler syndrome in which a dominant mutation in the COL2A1 gene has been identified. Linkage to COL2A1 was excluded in the two remaining families. The most striking difference between these two types of families was the absence of severe myopia and retinal detachment in the two unliked families. In the COL2A1 unlinked families, linkage of the phenotype to genes (COL11A1 and COL11A2) that encode pro alpha chains of type XI collagen, a minor cartilage-specific collagen, was also excluded. Since Stickler syndrome can be produced by mutations in COL2A1, COL11A1, and COL11A2, our data suggest that there is at least a fourth locus for Stickler syndrome.

Mosaic trisomy 16 ascertained through amniocentesis: evaluation of 11 new cases.

Trisomy 16, once thought to result uniformly in early pregnancy loss, has been detected in chorionic villus samples (CVS) from on-going pregnancies and was initially ascribed to a second, nonviable pregnancy. Prenatally detected trisomy 16 in CVS and its resolution to disomy has led to the reexamination of the viability of trisomy 16. This study evaluates 11 cases of mosaic trisomy 16 detected through second trimester amniocentesis. In 9 of the 11 cases, amniocenteses were performed in women under the age of 35 because of abnormal levels of maternal serum alpha-fetoprotein (MSAFP) or maternal serum human chorionic gonadotropin (MShCG). The other two amniocenteses were performed for advanced maternal age. Five of the 11 pregnancies resulted in liveborn infants, and six pregnancies were electively terminated. The liveborn infants all had some combination of intrauterine growth retardation (IUGR), congenital heart defects (CHD), or minor anomalies. Two of them died neonatally because of complications of severe congenital heart defects. The three surviving children have variable growth retardation, developmental delay, congenital anomalies, and/or minor anomalies. In the terminated pregnancies, the four fetuses evaluated by ultrasound or autopsy demonstrated various congenital anomalies and/or IUGR. Cytogenetic and fluorescent in situ hybridization studies identified true mosaicism in 5 of 10 cases examined, although the abnormal cell line was never seen in more than 1% of cultured lymphocytes. Placental mosaicism was seen in all placentas examined and was associated with IUGR in four of seven cases. Maternal uniparental disomy was identified in three cases. Mosaic trisomy 16 detected through amniocentesis is not a benign finding but associated with a high risk of abnormal outcome, most commonly IUGR, CHD, developmental delay, and minor anomalies. The various outcomes may reflect the diversity of mechanisms involved in the resolution of this abnormality. As 80% of these patients were ascertained because of the presence of abnormal levels of MSAFP or MShCG, the increased use of maternal serum screening should bring more such cases to clinical attention.