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.

Tetrasomy 12p--unusual presentation in CVS.

CVS direct preparations usually achieve limited resolution and are better at detecting numerical rather than structural abnormalities. A CVS direct preparation analyzed using G-banding revealed a 47,XY,+G karyotype in 5 of 11 cells and was reported as mosaic for trisomy 21. Subsequent analysis of the CVS culture found only normal male cells. Amniocentesis revealed both normal male cells and cells with an extra F-group chromosome. Fluorescence in situ hybridization (FISH) identified this chromosome to be an isochromosome from the short arm of chromosome 12 [i(12)(p10)]. The amniocyte karyotype was reported as 47,XY,+i(12)(p10)[12]/46,XY[8].ish i(12)(p10)(wcp12+), which is associated with Pallister-Killian syndrome. Reexamination of the CVS direct preparation by FISH with a chromosome 12 centromere probe confirmed the karyotype of this tissue to be 47,XY,+mar[5]/46,XY[6].nuc ish 12cen(D12Z3 x 3)/12cen(D12Z3 x 2). Thus, multiple studies, including amniocentesis and fluorescence in situ hybridization, may be required to fully and accurately evaluate abnormalities detected by CVS. This case also indicates that mosaicism for supernumerary isochromosomes may have a complex origin.

Corneal transplantation in a patient with mucopolysaccharidosis type VII (Sly disease).

PURPOSE: To illustrate a good visual outcome following penetrating keratoplasty in a patient with Sly disease, a rare mucopolysaccharidosis (MPS) caused by a deficiency of beta-glucuronidase. METHODS: A 15-year-old male with progressive bilateral corneal opacification had a complete medical, genetic, and ophthalmic evaluation followed by a penetrating keratoplasty. RESULTS: The cornea has remained clear for two years following surgery. Histopathology of the corneal button demonstrated vacuoles and granular inclusions consistent with this lysosomal storage disease. CONCLUSION: While research is ongoing in the fields of enzyme replacement and bone marrow transplantation, these treatments may not alleviate or reverse the corneal clouding. This case illustrates that cornea transplantation may be a valuable treatment option for visually rehabilitating such patients.

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.

Radical surgery for extensive rectal cancer: is it worthwhile?

In a small proportion of patients with extensive primary or locally recurrent rectal cancer, disease remains confined to the pelvis for a prolonged period. Symptoms are highly prejudicial to quality of life and often refractory to treatment short of extirpative surgery. Cure requires en bloc excision of all involved pelvic viscera with tumor-free margins. The pelvic exenterations (PE) are the most radical operations for rectal cancer. PE carries a high risk of perioperative morbidity and mortality, and has profound functional, psychological, and psychosexual implications for patients. Careful preoperative counseling regarding surgical risks and the impact of PE on body function and image is indispensable; the patient's consent must be truly informed. Patients with major medical or psychiatric/emotional comorbidity and those who are mentally incompetent are not candidates. Tenesmus and central pelvic/perineal pain are amenable to PE whereas radicular pain is not; sciatica and lower extremity lymphedema portend unresectability. Extrapelvic disease should be excluded preoperatively. While invaded sacrum can be resected en bloc with involved viscera (sacropelvic exenteration), fixity of tumor to the pelvic sidewall(s) in nonirradiated patients almost invariably implies unresectability. Other contraindications to PE include invasion of the proximal (S1 or higher) lumbosacral spine or lumbosacral plexus/sciatic nerves, ureteric obstruction proximal to the ureterovesical junctions, and encasement of the external or common iliac vessels by tumor. PE for advanced primary rectal carcinoma yields 5-year survival of over 40%; when performed for recurrent disease, long-term salvage rates are not as high. While radical surgery is rarely indicated for palliation, PE in carefully selected (good performance status and life expectancy, complete excision of all gross disease) incurable patients results in abrogation of disabling symptoms and reasonable intervals of high-quality survival.

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.

Mitochondrial gene mutation is a significant predisposing factor in aminoglycoside ototoxicity.

PURPOSE: Aminoglycoside-induced deafness caused by mutations in the mitochondrial 12S ribosomal RNA gene has been described in a number of Asian patients. The purpose of the current study is to analyze ethnically diverse patients in the United States with hearing loss after aminoglycoside exposure for presence or absence of these mitochondrial DNA mutations, and establish the frequency and clinical presentation associated with them. PATIENTS AND METHODS: Clinical histories, medical records, and blood samples were obtained from 41 unrelated American individuals with hearing loss after aminoglycoside exposure. DNA was extracted from the blood of these individuals, amplified by the polymerase chain reaction, and analyzed for mitochondrial ribosomal RNA gene mutations by allele-specific oligonucleotide hybridization, restriction fragment length polymorphism analysis, and sequencing. RESULTS: The nucleotide 1555 A-->G mutation was identified in 7 of 41 individuals (17%). None of the other known mutations was found. The ethnic origin of the individuals with predisposing mutations included Caucasians, Hispanics, and Asians. Four of the 7 patients with the 1555 A-->G mutation had a family history of aminoglycoside-induced ototoxicity. Particularly unexpected was the late onset of hearing loss in 3 of these patients, years after the aminoglycoside exposure. The 12S ribosomal RNA gene was sequenced in these patients, and a second sequence change that could be responsible for the milder phenotype was detected in 1 of the 3 patients. CONCLUSION: These findings imply that a significant proportion of patients with aminoglycoside-induced ototoxicity harbor mutations in the 12S rRNA gene, which can be detected by DNA screening. Also, the majority of these hearing losses could have been easily prevented by the simple taking of a clinical history. In these individuals, a genetic susceptibility to the ototoxic effects of aminoglycosides can be diagnosed, and deafness can be prevented in maternal relatives by avoidance of these antibiotics.

The ocular presentation of neurofibromatosis 2.

Neurofibromatosis 2 (NF2) is an inherited disorder characterised primarily by bilateral vestibular schwannomas and other central nervous system tumours. Individuals with NF2 also have early onset cortical and posterior subcapsular or capsular cataract and other ocular abnormalities, such as retinal hamartomas. Although their diagnostic significance is rarely appreciated, the ocular manifestations are often the first sign of disease. We describe 5 cases that illustrate the diverse ocular manifestations of NF2.

Involvement of CD44 and the cytoskeletal linker protein ankyrin in human neutrophil bacterial phagocytosis.

The leukocyte CD44 and CD45 cell surface receptors are associated via the linker proteins ankyrin and fodrin with the cytoskeleton, which itself is important in immune cell functions such as adherence, chemotaxis, and phagocytosis. The effects of rat antihuman CD44 and CD45 monoclonal antibodies on phagocytosis of fluoresceinated heat-killed Staphylococcus aureus 502A by normal human neutrophils (PMNs) during 2 hr incubation in RPMI-1640 was studied via flow cytometry and confocal microscopy. Flow cytometry was performed using an excitation wavelength of 488 nm, fluorescence being measured at 515-560 nm on 50,000 PMNs per sample. Confocal microscopy was performed on samples after further incubation with rhodamine-conjugated antiankyrin. Anti-CD44 resulted in an increase of 27-31% compared to control (P = 0.004) in the proportion of PMNs fluorescing, an increase of 17-24% (P = 0.001) in mean intracellular fluorescence per PMN, and an increase in total PMN fluorescence of 50-58% compared to control (P < 0.001). In contrast, anti-CD45 had little effect on phagocytosis. Colchicine (a microtubule-disrupting agent) enhanced, whereas cytochalasin-D (a microfilament inhibitor) inhibited bacterial phagocytosis; cytochalasin-D completely abrogated the effect of anti-CD44 on this PMN function. Hyaluronic acid augmented phagocytosis by an increment similar to that observed with anti-CD44. Two-color flow cytometry and confocal microscopy demonstrated that ankyrin always colocalized with ingested fluorescein isothiocyanate (FITC)-labeled bacteria. These data strongly suggest that CD44 is involved in bacterial phagocytosis, provide further evidence of CD44 receptor linkage to cytoskeletal elements in human leukocytes, and suggest that ankyrin has a significant role in the transport of phagosomes.

Translocation (12;15)(p13;q13) in a patient with relapsed acute myeloid leukemia.

We report a patient with a history of leukopenia who developed acute myeloid leukemia (AML) FAB M2 and was successfully treated with induction and consolidation chemotherapy. She relapsed 7 months after initial diagnosis. Peripheral blood cells at relapse showed a t(12;15)(p13;q13), which has not been previously described in de novo or relapsed AML.

Controversies in counseling for mitochondrial conditions.

A healthy woman sought preconceptional genetic counseling regarding a family history of a mitochondrial myopathy in her brother and retinitis pigmentosa (RP) in her two maternal aunts. Several questions were raised: (1) What is the likelihood of a familial mitochondrial condition? (2) What molecular tests or prenatal screening can we offer? (3) How would these tests help assess the likelihood of a familial mitochondrial condition? A mitochondrial mutation previously identified in the brother consisted of a heteroplasmic 2.9 kb deletion. We detected this deletion in the peripheral blood of the brother by PCR amplification of the deletion breakpoint, but not in his mother, the consultand, nor in one of the two aunts affected with RP. Although the molecular analysis was encouraging to the consultand, a familial mitochondrial disorder could not be eliminated with certainty. The pros and cons of prenatal testing for mitochondrial disorders are discussed in general, and as specifically related to this family.

Prenatal identification of i(Yp) by molecular cytogenetic analysis.

An i(Yp) is a rare marker chromosome. We present a case of de novo 46,X,i(Yp) detected prenatally in an amniotic fluid specimen. Fluorescence in situ hybridization (FISH) studies using a panel of Y-specific biotinylated DNA probes identified the marker chromosome as i(Yp). Comparative genomic hybridization (CGH) studies further confirmed the diagnosis. Upon pregnancy termination, external examination of the fetus revealed a generally well-developed male fetus with slight facial dysmorphism and prominent rocker-bottom feet. The molecular cytogenetic data in this case proved very useful in genetic counselling and served as a good example illustrating the important role of molecular techniques for accurate identification of marker chromosomes.

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.

Serum hyaluronate in the assessment of liver endothelial cell function after orthotopic liver transplantation in the rat.

This study was designed to evaluate the use of serum hyaluronate as a marker of liver endothelial cell function after liver transplantation. We performed orthotopic liver transplantation in both isogeneic and allogeneic rejector models. After transplantation, hepatocyte function was assessed on the basis of serum ALT and total bilirubin levels, and liver endothelial cell function was judged on the basis of serum hyaluronate levels. Significant increase of hyaluronate in the rejector model, compared with the isogeneic model, was seen before any significant results could be obtained from conventional liver function tests. The impaired metabolism of hyaluronate in the rejector model was observed after intravenous injection of trace amounts of radioactive material. Serial studies demonstrate that the endothelial cell is a more susceptible target for the immune response than the hepatocyte. Serum hyaluronate concentration may be a better indicator in the early assessment of graft function. We also examined serum hyaluronate levels to evaluate cold ischemia-reperfusion injury to the liver endothelial cells in the isogeneic model. At 2 hr after reperfusion, hyaluronate levels in the 6-hr cold ischemia (nonviable allograft) group were significantly higher than in the 1-hr and 3-hr cold ischemia (viable allograft) groups. However, there was little difference between the viable allograft groups. After an intravenous injection of 1 mg/kg hyaluronate, the hyaluronate elimination rate in the 3-hr group was distinctly slower than that in the 1-hr group. These data indicate that the hyaluronate elimination rate may be a more sensitive marker of liver endothelial cell function in viable liver after a short period of ischemia.

Pelvic exenteration for recurrent colorectal carcinoma: a review.

Pelvic recurrence following curative resection for colorectal carcinoma continues to pose a challenge to the oncologist despite current multimodality therapy. Pelvic exenteration with or without sacral resection may provide long-term disease-free survival and a chance of cure for a small subset of patients in whom the recurrent disease is confined to the pelvis and can be resected with "clear" margins. For others with residual disease, exenteration may offer good palliation for the intractable symptoms, but no survival advantage. The clinical decision to perform exenteration with palliative intent must be individualized. This is generally not advised because of the short life expectancy in the face of prolonged convalescence. This technically demanding procedure is associated with significant morbidity, especially in patients with prior pelvic radiation. Current advances in urinary diversion and methods of pelvic reconstruction may significantly reduce these problems. The surgeon's experience and careful patient selection remain the most important determinants of success with this operation.

Phloridzin and phloretin inhibition of 2-deoxy-D-glucose uptake by tumor cells in vitro and in vivo.

Utilizing the rat mammary adenocarcinoma and Fischer bladder cell carcinoma cell lines, this study demonstrated the ability of two known glucose transport inhibitors, phloridzin (P1) and its aglucone, phloretin (P2), to block glucose transport into whole viable tumor cells in vitro and tumor tissues in vivo. This work represents the first in a series of experiments designed to explore the efficacy of P1 and P2 administration in restraining tumor cell growth via the inhibition of glucose transmembrane transport.

The efficacy of phloridzin and phloretin on tumor cell growth.

This study utilized phloridzin (P1) and its aglucone phloretin (P2), two known inhibitors of glucose transmembrane transport, to inhibit tumor cell growth in vivo. The efficacy of hydrazine sulfate as an anticachexic agent was also evaluated. Utilizing the rat mammary adenocarcinoma and Fischer bladder cell carcinoma cell lines, it has been shown that the i.p. administration of P1 and P2 can produce significant differences in mean tumor diameters as compared to the untreated controls.