Carlos Eduardo Steiner

Mutations in the gene encoding filamin B disrupt vertebral segmentation, joint formation and skeletogenesis

The filamins are cytoplasmic proteins that regulate the structure and activity of the cytoskeleton by cross-linking actin into three-dimensional networks, linking the cell membrane to the cytoskeleton and serving as scaffolds on which intracellular signaling and protein trafficking pathways are organized (reviewed in refs. 1,2). We identified mutations in the gene encoding filamin B in four human skeletal disorders. We found homozygosity or compound heterozygosity with respect to stop-codon mutations in autosomal recessive spondylocarpotarsal syndrome (SCT, OMIM 272460) and missense mutations in individuals with autosomal dominant Larsen syndrome (OMIM 150250) and the perinatal lethal atelosteogenesis I and III phenotypes (AOI, OMIM 108720; AOIII, OMIM 108721). We found that filamin B is expressed in human growth plate chondrocytes and in the developing vertebral bodies in the mouse. These data indicate an unexpected role in vertebral segmentation, joint formation and endochondral ossification for this ubiquitously expressed cytoskeletal protein.

Gray and white matter imbalance – Typical structural abnormality underlying classic autism?

Recent evidence supports increased cortical activity and impaired brain connectivity in autism, but the structural correlates of these abnormalities are not yet defined. We performed a voxel based morphometry analysis of brain MRI from patients with autism selected from a group of 103 subjects with pervasive developmental disorders. Twelve male patients with mean age of 12.4 +/- 4 years were compared with 16 matched controls. Patients with autism exhibited increase in gray matter in medial and dorsolateral frontal areas, in the lateral and medial parts of the temporal lobes, in the parietal lobes, cerebellum and claustrum. Patients also showed decrease in frontal, parietal, temporal and occipital white matter. The combination of enlarged cortex and reduced white matter is possibly the structural basis of some symptoms of classic autism.

Mucopolysaccharidoses in Brazil: what happens from birth to biochemical diagnosis?

Mucopolysaccharidoses (MPS) form a group of inherited metabolic disorders characterized by intralysosomal storage of glycosaminoglycans. This study aimed to investigate the path followed by Brazilian patients from birth to diagnosis. An interview was conducted with patients parents or guardians with subsequent review of patients medical records. One hundred thirteen patients with MPS were included (MPS I: 18, MPS II: 43, MPS IIIA: 2, MPS IIIB: 3, MPS IIIC: 1, MPS IVA: 15, MPS IVB: 1, MPS VI: 29, MPS VII: 1) from 97 families. Median age at the onset of signs/symptoms was 18 months (MPS I: 18, MPS II: 24, MPS IVA: 8, MPS VI: 8). Skeletal abnormalities (for MPS IVA and MPS VI), joint contractures (for MPS II), and typical facial features (for MPS I) were the most frequently reported first signs/symptoms. Several health professionals were involved in patients care as of the onset of symptoms until biochemical diagnosis was established. Median age at diagnosis was 76 months (MPS I: 75, MPS II: 95, MPS IVA: 75, MPS VI: 52). Considering the group as a whole, there was a 4.8‐year delay between the onset of signs/symptoms and the establishment of the diagnosis. Considering that specific therapies are available for some of these disorders and that early treatment is likely to change more favorably the natural history of the disease, efforts should be made to minimize this delay. We believe that this situation can be improved by measures that both increase awareness of health professionals about MPS and improve access to diagnostic tests.

Genotypic and phenotypic analysis of 396 individuals with mutations in Sonic Hedgehog

Background Holoprosencephaly (HPE), the most common malformation of the human forebrain, may result from mutations in over 12 genes. Sonic Hedgehog (SHH) was the first such gene discovered; mutations in SHH remain the most common cause of non-chromosomal HPE. The severity spectrum is wide, ranging from incompatibility with extrauterine life to isolated midline facial differences. Objective To characterise genetic and clinical findings in individuals with SHH mutations. Methods Through the National Institutes of Health and collaborating centres, DNA from approximately 2000 individuals with HPE spectrum disorders were analysed for SHH variations. Clinical details were examined and combined with published cases. Results This study describes 396 individuals, representing 157 unrelated kindreds, with SHH mutations; 141 (36%) have not been previously reported. SHH mutations more commonly resulted in non-HPE (64%) than frank HPE (36%), and non-HPE was significantly more common in patients with SHH than in those with mutations in the other common HPE related genes (p<0.0001 compared to ZIC2 or SIX3). Individuals with truncating mutations were significantly more likely to have frank HPE than those with non-truncating mutations (49% vs 35%, respectively; p=0.012). While mutations were significantly more common in the N-terminus than in the C-terminus (including accounting for the relative size of the coding regions, p=0.00010), no specific genotype―phenotype correlations could be established regarding mutation location. Conclusions SHH mutations overall result in milder disease than mutations in other common HPE related genes. HPE is more frequent in individuals with truncating mutations, but clinical predictions at the individual level remain elusive.

Frequency of the different mutations causing spinocerebellar ataxia (SCA1, SCA2, MJD/SCA3 and DRPLA) in a large group of Brazilian patients

Spinocerebellar ataxia type 1 (SCA1), spinocerebellar ataxia type 2 (SCA2) and Machado-Joseph disease or spinocerebellar ataxia type 3 (MJD/SCA3) are three distinctive forms of autosomal dominant spinocerebellar ataxia (SCA) caused by expansions of an unstable CAG repeat localized in the coding region of the causative genes. Another related disease, dentatorubropallidoluysian atrophy (DRPLA) is also caused by an unstable triplet repeat and can present as SCA in late onset patients. We investigated the frequency of the SCA1, SCA2, MJD/SCA3 and DRPLA mutations in 328 Brazilian patients with SCA, belonging to 90 unrelated families with various patterns of inheritance and originating in different geographic regions of Brazil. We found mutations in 35 families (39%), 32 of them with a clear autosomal dominant inheritance. The frequency of the SCA1 mutation was 3% of all patients; and 6% in the dominantly inherited SCAs. We identified the SCA2 mutation in 6% of all families and in 9% of the families with autosomal dominant inheritance. The MJD/SCA3 mutation was detected in 30% of all patients; and in the 44% of the dominantly inherited cases. We found no DRPLA mutation. In addition, we observed variability in the frequency of the different mutations according to geographic origin of the patients, which is probably related to the distinct colonization of different parts of Brazil. These results suggest that SCA may be occasionally caused by the SCA1 and SCA2 mutations in the Brazilian population, and that the MJD/SCA3 mutation is the most common cause of dominantly inherited SCA in Brazil.

Molecular genetics study of deafness in Brazil: 8-year experience.

Hereditary hearing loss is a complex disorder that involves a large number of genes. In developed countries, 1 in 1,000 children is born with deafness severe enough to require special education services, and about 60% of the cases of isolated deafness have a genetic origin. Although more than 100 genes for hearing loss are known currently, only a few are routinely tested in the clinical practice. In this study, we present our findings from the molecular diagnostic screening of the GJB2 and GJB3 genes, del(GJB6‐D13S1830) and del(GJB6‐D13S1854) deletions in the GJB6 gene, Q829X mutation in the otoferlin gene (OTOF) and, the A1555G and A7445G mutations in the mitochondrial genome over an 8‐year period. Mutations analysis in the previously mentioned genes and mutations was performed on 645 unrelated Brazilian patients with hearing loss who fell into two different testing groups. Different mutations in the GJB2 gene were responsible for most of cases studied, but deletions in the GJB6 gene as well as mitochondrial mutations were also found. While most cases of hearing loss in this country are due to environmental factors, the genetic etiology of deafness will increasingly be determined as more genetic tests become available.

G59S mutation in the GJB2 (connexin 26) gene in a patient with Bart–Pumphrey syndrome

The connexins are a family of proteins whose major function is as part of the gap junctions of cell-to-cell channels. They are expressed in several tissues including brain, skin, and cochlea. Mutations in connexins play a major role in nonsyndromic deafness, but have been also described in individuals with variable dermatological features [Kelsell et al., 2001]. Initially, the connexin 26 (GJB2) gene was implicated in autosomal recessive forms of hearing loss, but was later found to be involved in both recessive and dominant forms of genetic deafness [Chaib et al., 1994; Petit et al., 2001]. The most common mutation associated with recessive deafness is 35delG within the GJB2 gene [Denoyelle et al., 1997], which accounts for up 70% of the connexin 26 mutations observed in European and American Caucasian populations [Green et al., 1999; Gasparini et al., 2000]. In addition, there are other mutations such as nonsense mutations, deletions, and insertions within the GJB2 gene that result in hearing loss. Heterozygous missense mutations in GJB2 were found in several conditions associating deafness and hyperkeratosis: D66H in mutilating keratoderma with sensorineural deafness (Vohwinkel’s syndrome, OMIM 124500) [Maestrini et al., 2002], G59A and R75Q in palmoplantar hyperkeratosis with deafness (PPKD, OMIM 148350) [Heathcote et al., 2000; Uyguner et al., 2002], as well as D50Y and D50N in keratitisichthyosis-deafness syndrome (KID, OMIM 148210), and in hystrix like-ichthyosis-deafness syndrome (HID, OMIM 602540) [Richard et al., 2002; van Geel et al., 2002; Yotsumoto et al., 2003]. Richard et al. [2002] also described two sporadic cases of KID syndrome with G12R and S17F missense mutations, each patient showing a different mutation. GJB2 is contiguous to another connexin gene, GJB6, which encodes connexin 30. Together they form the DFBN1 locus. Large deletions in GJB6 extending to the GJB2 gene may result in a monogenic or digenic pattern of prelingual deafness. Missense mutations in the GJB6 gene cause hidrotic ectodermal dysplasia (Clouston syndrome), an autosomal dominant disorder characterized by hair and nail defects, palmoplantar hyperkeratosis, and other variable findings. Mutations in the GJB3 (connexin 31) gene also cause both autosomal recessive and autosomal dominant forms of nonsyndromic deafness, as well as erythrokeratodermia variabilis, an autosomal dominant genodermatosis with variable clinical presentation including hyperkeratosis. Bart–Pumphrey syndrome (BPS, OMIM 149200) is a rare autosomaldominantdisorder characterizedbycongenitaldeafness, palmoplantar hyperkeratosis, knuckle pads, and leukonychia. To date, only a few families with this disorder have been reported [Schwann, 1963; Bart and Pumphrey, 1967; Crosbyand Vidurrizaga,1976; Ramer etal., 1994; Oliveiraetal., 2003]. Richard et al. [2004] recently reported BPS mutation. We present here a 26-year-old male patient with BPS who was submitted to molecular analysis of the connexin 26, 30, and 31 genes. He presented with congenital sensorineural deafness. Verrucous-like nodes on the interphalangeal, metacarpophalangeal, and metatarsophalangeal joints of the dorsa of the hands and feet (knuckle pads, Fig. 1) as well as palmoplantar hyperkeratosis started at age 3 years. Nail shape and size were normal, and no leukonychia was observed. Anthropometrical data were within normal ranges and no signs of cognitive impairment were detected. An audiogram and a brainstem evoked response audiometry (BERA) were performed, both showing severe sensorineural hearing loss. His father showed a similar phenotype. Clinical presentation and hystologic findings of this family were previously reported by Oliveira et al. [2003]. Since the father was not willing to participate in molecular studies, blood specimens were collected only from the propositus with approval by the appropriate institutional review board and informed consent of the subject. DNA samples were extracted from whole blood by standard techniques. The GJB2 gene was PCR amplified using the two pairs of primers [Denoyelle et al., 1997; Kelsell et al., 1997], and GJB3 gene was amplified using five pairs of primers described elsewhere [Xia et al., 1998]. For mutation analysis, the single coding regions of the GJB2 and GJB3 genes were sequenced from PCR products using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (ABI PRISM/PE Biosystems, Foster City, CA) and the products resolved on ABI PRISM 377 (Perkin Elmer, Boston, MA). In order to examine the deletion of the GJB6 gene, we amplified the breakpoint junction of the (GJB6-D13S1830)del by PCR [del Castillo et al., 2002]. Analysis and sequencing of GJB3 did not show any abnormality and the (GJB6-D13S1830)del in the GJB6 gene was not found. However, sequencing of GJB2 revealed a G to A substitution at nucleotide 175. This nucleotide substitution corresponds to a heterozygous glycine to serine change at codon 59 (G59S) (Fig. 2). This amino acid substitution was not found in more than 200 individuals with isolated deafness and in over 100 individuals without hearing loss tested in our service. This substitution was also not listed in the Human Gene Mutation Database [2004]. A mutation in the same codon (59) was reported by Heathcote et al. [2000] who proposed that G59A was responsible for the syndrome of hearing loss and hyperkeratosis observed in their study. This codon is located in the first extracellular loop (E1) of connexin 26 (Fig. 3), a region that is highly conserved among all connexins sequenced to date. Most syndromic forms of deafness-hyperkeratosis are associated with mutations in the first extracellular domain of Grant sponsor: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).

Genetic and neurological evaluation in a sample of individuals with pervasive developmental disorders

With the aim of analyzing which complementary tests are relevant in the diagnostic evaluation of individuals with pervasive developmental disorders, a protocol of clinical and laboratory evaluation was applied in 103 outpatients. The protocol included chromosomal analysis, screening for inborn errors of metabolism, cytogenetic and molecular study of the FRAXA, FRAXE, and FRAXF mutations, EEG, SPECT, and magnetic resonance imaging study. Eighty-four subjects concluded the complementary tests and were classified either as having autism, atypical autism or Asperger syndrome according to the DSM-IV criteria. Sixteen individuals, all bellonging to the two autistic groups, presented genetic or enviromental factors that may have lead to the behavioral disorders, showing the importance of diagnostic evaluation in this group of conditions. Neuroimaging and EEG findings were non-specific and occurred in similar proportion among the groups, being considered of relative low significance in the diagnostic evaluation of individuals with pervasive developmental disorders.

Additional EFNB1 mutations in craniofrontonasal syndrome

Deeann Wallis, Felicitas Lacbawan, Mahim Jain, Vazken M. Der Kaloustian, Carlos E. Steiner, John B. Moeschler, H. Wolfgang Losken, Ilkka I. Kaitila, Stephen Cantrell, Virginia K. Proud, John C. Carey, Donald W. Day, Dorit Lev, Ahmad S. Teebi, Luther K. Robinson, H. Eugene Hoyme, Nadia Al-Torki, Jacqueline Siegel-Bartelt, John B. Mulliken, Nathaniel H. Robin, Dolores Saavedra, Elaine H. Zackai, and Maximilian Muenke* Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland McGill University Health Center, Montreal, Canada Department of Medical Genetics, Unicamp, Campinas, Sao Paulo, Brazil Dartmouth Medical Center, Lebanon, New Hampshire University of Pittsburgh, Pittsburgh, Pennsylvania Haartman Institute, University of Helsinki, Helsinki, Finland New Jersey Dental School, Newark, New Jersey Children’s Hospital of the King’s Daughters, Norfolk, Virginia University of Utah Medical Center, Salt Lake City, Utah Private Practice, Hewitt, Texas Institute of Clinical Genetics, Wolfson Medical Genetics Center, Holon, Israel Weill Cornell Medical College, New York, New York Buffalo School of Medicine and Biomedical Sciences, Buffalo, New York Stanford University School of Medicine, Stanford, California Kuwait Medical Genetics Center, Sulibihkhat, Kuwait The Genetics Institute, Pasadena, California Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts University of Alabama, Birmingham, Alabama Hospital General Dr. Manuel Gea Gonzáles, Mexico City, Mexico Division of Human and Molecular Genetics, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania

Spondylocarpotarsal synostosis with ocular findings

We report on three sibs presenting with spondylocarpotarsal synostosis, short-trunk dwarfism of postnatal onset, scoliosis, unsegmented thoracic vertebrae with unilateral bar, and carpal bone fusion. Tarsal bone fusion and dental abnormalities were noted in some of them, indicating pleiotropy and intrafamilial variability. Lens opacities, rarefaction of retinal pigmentation, and narrowing of retinal vessels, detected in two patients, are findings that have not been described to date in this condition.