Mutations in FLNB cause boomerang dysplasia.

Boomerang dysplasia (BD) is a perinatal lethal osteochondrodysplasia, characterised by absence or underossification of the limb bones and vertebrae. The BD phenotype is similar to a group of disorders including atelosteogenesis I, atelosteogenesis III, and dominantly inherited Larsen syndrome that we have recently shown to be associated with mutations in FLNB, the gene encoding the actin binding cytoskeletal protein, filamin B. We report the identification of mutations in FLNB in two unrelated individuals with boomerang dysplasia. The resultant substitutions, L171R and S235P, lie within the calponin homology 2 region of the actin binding domain of filamin B and occur at sites that are evolutionarily well conserved. These findings expand the phenotypic spectrum resulting from mutations in FLNB and underline the central role this protein plays during skeletogenesis in humans.

AT-rich palindromes mediate the constitutional t(11;22) translocation.

The constitutional t(11;22) translocation is the only known recurrent non-Robertsonian translocation in humans. Offspring are susceptible to der(22) syndrome, a severe congenital anomaly disorder caused by 3&rcolon;1 meiotic nondisjunction events. We previously localized the t(11;22) translocation breakpoint to a region on 22q11 within a low-copy repeat termed "LCR22" and within an AT-rich repeat on 11q23. The LCR22s are implicated in mediating different rearrangements on 22q11, leading to velocardiofacial syndrome/DiGeorge syndrome and cat-eye syndrome by homologous recombination mechanisms. The LCR22s contain AT-rich repetitive sequences, suggesting that such repeats may mediate the t(11;22) translocation. To determine the molecular basis of the translocation, we cloned and sequenced the t(11;22) breakpoint in the derivative 11 and 22 chromosomes in 13 unrelated carriers, including two de novo cases and der(22) syndrome offspring. We found that, in all cases examined, the reciprocal exchange occurred between similar AT-rich repeats on both chromosomes 11q23 and 22q11. To understand the mechanism, we examined the sequence of the breakpoint intervals in the derivative chromosomes and compared this with the deduced normal chromosomal sequence. A palindromic AT-rich sequence with a near-perfect hairpin could form, by intrastrand base-pairing, on the parental chromosomes. The sequence of the breakpoint junction in both derivatives indicates that the exchange events occurred at the center of symmetry of the palindromes, and this resulted in small, overlapping staggered deletions in this region among the different carriers. On the basis of previous studies performed in diverse organisms, we hypothesize that double-strand breaks may occur in the center of the palindrome, the tip of the putative hairpin, leading to illegitimate recombination events between similar AT-rich sequences on chromosomes 11 and 22, resulting in deletions and loss of the palindrome, which then could stabilize the DNA structure.

Stroke in children: genetic and metabolic issues.

The study of genetic and metabolic etiologies of pediatric stroke, both vascular and metabolic, allows an understanding of the causes of acute focal neurologic deficits in childhood. Here, the mendelian and mitochondrial genetic causes of pediatric stroke syndromes are reviewed. This approach elucidates the etiology of childhood stroke and illustrates many of the genetic risk factors that are found in adult-onset cerebrovascular disease. Therefore, the study of childhood stroke serves as a model to elucidate the potential risk factors for all stroke. Ultimately this will serve to develop a more rational preventive and therapeutic approach for all cerebrovascular disease.

Inherited duplication Xq27-qter at Xp22.3 in severely affected males: molecular cytogenetic evaluation and clinical description in three unrelated families.

We describe the clinical phenotype in four males from three families with duplication (X)(qter-->q27::p22.3-->qter). This is an unusual duplication of the distal long arm segment, Xq27-qter, onto the distal short arm of the X chromosome at Xp22.3, as shown by fluorescent in situ hybridization analysis with multiple X-specific probes. The patients are young male offspring of three unrelated, phenotypically normal carrier women. The affected males have similar clinical manifestations including severe growth retardation and developmental delay, severe axial hypotonia, and minor anomalies. Such clinical similarity in three unrelated families demonstrates that this chromosome abnormality results in a new and distinct clinical phenotype. Replication studies, performed on two of the mothers, provided evidence that inactivation of the abnormal X chromosome permitted the structural abnormality to persist in these families for a generation or more in females without phenotypic expression.

Subclavian artery pseudoaneurysm in type IV Ehlers-Danlos syndrome.

We report case of a subclavian artery pseudoaneurysm in a patient with type IV Ehlers-Danlos Syndrome. A 16-year-old boy underwent successful repair of a subclavian artery pseudoaneurysm that occurred after a cervical hyperextension injury. Subsequent workup included skin biopsy and fibroblast culture, which were consistent with a diagnosis of type IV Ehlers-Danlos Syndrome. This condition is a dominantly inherited connective tissue disorder, which in this patient was found to be caused by a spontaneous point mutation in the COL3A1 gene that encodes the chains of type III procollagen. The clinical, genetic, and molecular characteristics of type IV Ehlers-Danlos Syndrome are briefly reviewed.

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.

Tissue differences in fragile X mosaics: mosaicism in blood cells may differ greatly from skin.

The fragile X mutation is diagnosed from the structure of the FMR1 gene in blood cell DNA. An estimated 12 to 41% of affected males are mosaics who carry both a "full mutation" allele from which there is no gene expression and a "premutation" allele which has normal gene expression. We compared the DNA in blood cells and skin fibroblasts from four mosaic fragile X males to see if there was a difference in the relative amounts of premutation and full mutation alleles within the tissues of these individuals. Two of these males showed striking differences in the ratio of premutation to full mutation in different tissues while the other two showed only slight differences. These observations conform with the widely accepted hypothesis that the fragile X CGG repeat is unstable in somatic tissue during early embryogenesis. Accordingly, the mosaicism in brain and skin, which are both ectodermal in origin, may be similar to each other but different from blood which is not ectodermal in origin. Thus, the ratio of full mutation to premutation allele in skin fibroblasts might be a better indicator of psychological impairment than the ratio in blood cells.

Prenatal diagnosis of mitochondrial fatty acid oxidation defects.

Amniocytes isolated from two pregnancies at risk for fatty acid oxidation defects were incubated with stable isotopically labelled palmitate, in the presence of L-carnitine, to probe that pathway. The labelled acylcarnitines were then quantitated using tandem mass spectrometry. Amniocytes from a pregnancy at risk for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency produced a characteristic acylcarnitine profile with increased levels of octanoylcarnitine and decanoylcarnitine, indicative of MCAD deficiency. DNA analysis confirmed that the fetus was homozygous for the MCAD A985G mutation. Acylcarnitine and DNA analysis of the infant's blood obtained post-partum confirmed MCAD deficiency. Amniocytes from a pregnancy at risk for an unspecified fat oxidation defect produced increased levels of long-chain acylcarnitines consistent with a deficiency in very-long-chain acyl-CoA dehydrogenase (VLCAD). Measurements of the enzymatic activity confirmed VLCAD deficiency in amniocytes. Acylcarnitine profiles of the infant's blood obtained post-partum in addition to enzyme activities measured in fibroblasts confirmed VLCAD deficiency. The successful prenatal diagnosis of VLCAD and MCAD deficiencies using in vitro probes of fatty acid oxidation in fibroblasts suggests that this approach can potentially recognize many mitochondrial fatty acid oxidation defects even if no prior diagnosis is determined in the family at risk.

Characterization of a de novo t(Y;9) (q11.2;q22) by FISH technique.

A new case with a de novo translocation involving the long arms of chromosomes Y and 9 i.e. 46, X, t(Y;9) (q11.2;q22) was noted in a phenotypically normal male as revealed by GTG technique and subsequently confirmed using dual color whole chromosome painting probes. Since the Yq11.2 region was involved in the translocation the fecundity status of the individual has been questioned. Previous cases with (Y;autosomal) translocations have produced males with azoospermia, a variety of male gamete deformities and idiopathic sterility. The azoospermic factor (AZF) on the distal Yq11 band, which apparently controls spermatogenesis, may have been adversely affected due to a microdeletion, rearrangement or complete loss as a result of the translocation mechanism. Testicular histological and fertility tests of the proband at a postpubescent age will reveal the status. The sex determining region (SRY) on the Yp, which has been equated with the testis determining factor (TDF), is believed to be intact. Dual color whole chromosome painting probes, which served as an important adjunct to GTG banding, increased the degree of certainty for the characterization of the involved translocation. A concise review of balanced (Y ; autosome) translocation cases are annotated here.

Mitochondrial encephalomyopathy associated with a single nucleotide pair deletion in the mitochondrial tRNALeu(UUR) gene.

The investigation of pathogenic mitochondrial DNA (mtDNA) mutations has revealed a complex relation between patient genotype and phenotype. For unknown reasons, some mtDNA mutations produce specific clinical manifestations such as chronic progressive external ophthalmoplegia; myoclonic epilepsy and ragged-red fiber disease (MERRF); and mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS). To enhance our understanding of the association between genotype and phenotype, we investigated a patient with mitochondrial encephalomyopathy and severe cerebral calcifications for a mtDNA mutation. There was a deletion of one of three T:A nucleotide pairs in the tRNALeu(UUR) gene of the mtDNA involving positions 3271 to 3273. Pedigree analysis suggested that this mutation may have occurred spontaneously in the proband. This analysis represents the smallest mtDNA deletion observed to date and is the first deletion identified within a mitochondrial tRNA. This observation emphasizes the importance of delineating the precise mutation responsible for an oxidative phosphorylation disease for patient diagnosis as well as for genetic counseling of maternal lineage relatives.

Mechanisms of the origin of a G-positive band within the secondary constriction region of human chromosome 9.

We report on a so-called rare variant where a G-positive band was sandwiched within the secondary constriction (qh) region of chromosome 9 and is apparently different from previous cases when characterized by the fluorescence in situ hybridization technique. The major differences included duplication of beta-satellite and satellite III DNA sequences and bands 9q13-->q21.1, without duplication or inversion of the alphoid sequences. Based on the reported cases, at least four types of variations can be accounted for. A variety of mechanisms have been proposed to describe the origin of a G-positive band within the 9qh region, which appears to be similar when studied by routine cytogenetic techniques but differs by molecular methods. It is hypothesized that the clinical consequences depend upon the size of the G-positive band(s) duplicated, and a genetic inactivation mechanism might have some sort of influence during the so-called heterochromatinization process. It appears that heterochromatin, once thought to be composed of junk DNA, may have some role after all in suppression of gene(s) and/or spreading of inactivation, if genes are embedded within the heterochromatic region. Apparently, the mixture of different types of DNA creating patches of genetic debris have become a fundamental hidden treasure, where genetically active chromatin could be inactivated without dire consequences. The variable nature of heterochromatin has resulted in cytogenetic heteromorphisms of a number of human chromosomes. Their characterization by molecular techniques is becoming imperative, because fetal wastage have occurred in many situations where variant chromosomes were wrongly identified as chromosomal abnormalities.

Postnatal confirmation of prenatally diagnosed trisomy 16 mosaicism in two phenotypically abnormal liveborns.

Two phenotypically abnormal liveborns in whom trisomy 16 mosaicism was diagnosed prenatally by amniocentesis are described. Analysis of a percutaneous umbilical blood sample in one case revealed a normal chromosomal complement. Ultrasound examinations performed at the time of amniocentesis were normal. Serial sonography during the late second and third trimesters demonstrated progressive intrauterine growth retardation (IUGR) in both fetuses and a cardiac defect in one. At birth, both infants had dysmorphic features and multiple congenital anomalies. Trisomy 16 mosaicism was confirmed postnatally in both infants in skin fibroblasts; however, peripheral blood samples contained only chromosomally normal cells. The two mosaic trisomy 16 cases described in this report, together with the five confirmed cases reported previously, demonstrate the need for caution in the counselling of patients when trisomy 16 mosaicism is diagnosed prenatally in amniotic fluid samples. Such cases potentially can result in the birth of dysmorphic infants with significant birth defects, growth retardation, and possible developmental disabilities.

Allan-Herndon-Dudley syndrome: clinical and linkage studies on a second family.

We restudied a family with X-linked mental retardation (XLMR) originally reported in abstract form by Davis et al. [1981]. All 8 living affected males were examined. Characteristics included severe mental retardation, spastic paraplegia, dysarthria, muscle wasting, scoliosis, broad shallow pectus excavatum, long face, large ears with minor modeling anomalies, foot deformities, joint contractures, and neck drop. Stature, OFC, testicular volume, high resolution chromosome and fragile X studies, and plasma amino acids were all normal. Their manifestations closely resemble those of a large family with XLMR originally reported by Allan et al. [1944] and restudied by Stevenson et al. [1990]. This condition has been termed the Allan-Herndon-Dudley syndrome (AHDS). As AHDS has been mapped to Xq21, mapping studies were undertaken to determine if this family maps to the same location. These studies demonstrate tight linkage to Xq21, with a maximum lod score of 2.88 obtained with probe pX65H7 (DXS72). Multipoint analysis located the mutant gene quite close to pX65H7 (multipoint Z = 4.14), slightly more proximal in Xq21 than was suggested by the data from the original AHDS family. It appears likely that this family is the second reported family with AHDS.

Progression of cardiac disease in Emery-Dreifuss muscular dystrophy.

Emery-Dreifuss muscular dystrophy (EDMD) is a rare X-linked muscular dystrophy characterized by early contractures, progressive muscle weakness, and atrial arrhythmias. Recent reports suggest that there may be additional cardiac problems in affected males and that carrier females may also show ECG abnormalities. We restudied two large families with EDMD in order to determine the extent of these problems. We examined 10 affected males and interviewed 2 others. The 3 affected males less than 20 years old had no ECG changes. All affected men of 35 years or older had arrhythmias. One had more severe arrhythmias when asleep, indicating the usefulness of continuous 24-h ECG monitoring in the evaluation of males affected with EDMD. Two required pacemakers, 4 had already had a pacemaker placed, and 4 other affected men with pacemakers had died prior to this study. One affected man with a pacemaker developed ventricular bigeminy and another developed congestive heart failure. Thus of 10 affected males with pacemakers, 6 had additional cardiac symptoms and 4 have died. Males with EDMD may survive longer with a ventricular pacemaker, but this may increase the likelihood that they will develop cardiomyopathy and ventricular arrhythmias. Of 34 carrier females examined, 6 had arrhythmias typical of EDMD. Two required a pacemaker. The risk of arrhythmia increased with age. Results from one family should be extrapolated to another with caution, as there appears to be significant interfamilial variation. We suggest careful cardiologic follow-up of EDMD patients and regular cardiac evaluations for older carrier females.

Muscle enzymes and isoenzymes in Emery-Dreifuss muscular dystrophy.

Emery-Dreifuss muscular dystrophy (EDMD) is a rare X-linked muscular dystrophy. Creatine kinase (CK) activity usually is increased in serum of affected males, but results for aldolase and lactate dehydrogenase (LD) in serum have been inconsistent, as have those for CK in carrier females. There have been few studies of CK-MB or LD isoenzyme-1 (LD-1) in EDMD. We measured CK, CK-MB, LD, LD-1, and aldolase activity in sera of 84 members of two large families with EDMD. DNA analysis had been carried out on all subjects. Although CK, LD, and aldolase activities were significantly increased in affected males, CK activity was the most consistently increased and was the least subject to artifactual increases. Mean CK-MB in serum was mildly increased, but LD-1 was within the normal reference interval, suggesting that CK-MB is increased in skeletal muscle in EDMD, as has been found in other forms of dystrophy. CK decreased with age in affected males. We saw no significant increases of muscle enzymes or isoenzymes in 33 EDMD carriers studied, of whom 19 were obligate carriers and 14 had been identified by DNA analysis.

Apparent Ruvalcaba syndrome with genitourinary abnormalities.

The Ruvalcaba syndrome is a rare malformation syndrome characterized by skeletal dysplasia, facial anomalies, and mental retardation. We report on a 22-year-old woman with severe growth and mental retardation and numerous manifestations characteristic of the Ruvalcaba syndrome. In addition, she has several anomalies not previously described in the Ruvalcaba syndrome, including upslanting palpebral fissures, torus palatinus, hiatal hernia with gastroesophageal reflux, recurrent respiratory infections, pectus excavatum, equinovarous deformity, hypotonia, unilateral renal hypoplasia, an accessory ovary, and atretic fallopian tube. Review of published reports of Ruvalcaba syndrome confirms variability of the clinical and radiographic changes. Findings present in at least 50% of reported patients include mental retardation, short stature, pubertal delay, an abnormal nose (usually beaked) with hypoplastic nasal alae, microstomia with narrow maxilla, thin upper lip vermilion, broad hips, small hands, joint limitation, short fingers and toes, and vertebral abnormalities. Because 5 of the reported patients had renal abnormalities, a renal ultrasound or contrast study is indicated in the evaluation of these patients. Additional reports, particular from multiplex families, will be important to better characterize this syndrome.