Alan Shanske

TBX5 genetic testing validates strict clinical criteria for Holt-Oram syndrome

Holt-Oram syndrome (HOS) is an autosomal dominant heart-hand syndrome characterized by congenital heart disease (CHD) and upper limb deformity, and caused by mutations in the TBX5 gene. To date, the sensitivity of TBX5 genetic testing for HOS has been unclear. We now report mutational analyses of a nongenetically selected population of 54 unrelated individuals who were consecutively referred to our center with a clinical diagnosis of HOS. TBX5 mutational analyses were performed in all individuals, and clinical histories and findings were reviewed for each patient without reference to the genotypes. Twenty-six percent of the complete cohort was shown to have mutations of the TBX5 gene. However, among those subjects for whom clinical review demonstrated that their presentations met strict diagnostic criteria for HOS, TBX5 mutations were identified in 74%. No mutations were identified in those subjects who did not meet these criteria. Thus, these studies validate our clinical diagnostic criteria for HOS including an absolute requirement for preaxial radial ray upper limb malformation. Accordingly, TBX5 genotyping has high sensitivity and specificity for HOS if stringent diagnostic criteria are used in assigning the clinical diagnosis.

Muenke syndrome (FGFR3-related craniosynostosis): expansion of the phenotype and review of the literature.

Muenke syndrome is an autosomal dominant disorder characterized by coronal suture craniosynostosis, hearing loss, developmental delay, carpal and tarsal fusions, and the presence of the Pro250Arg mutation in the FGFR3 gene. Reduced penetrance and variable expressivity contribute to the wide spectrum of clinical findings in Muenke syndrome. To better define the clinical features of this syndrome, we initiated a study of the natural history of Muenke syndrome. To date, we have conducted a standardized evaluation of nine patients with a confirmed Pro250Arg mutation in FGFR3. We reviewed audiograms from an additional 13 patients with Muenke syndrome. A majority of the patients (95%) demonstrated a mild‐to‐moderate, low frequency sensorineural hearing loss. This pattern of hearing loss was not previously recognized as characteristic of Muenke syndrome. We also report on feeding and swallowing difficulties in children with Muenke syndrome. Combining 312 reported cases of Muenke syndrome with data from the nine NIH patients, we found that females with the Pro250Arg mutation were significantly more likely to be reported with craniosynostosis than males (P < 0.01). Based on our findings, we propose that the clinical management should include audiometric and developmental assessment in addition to standard clinical care and appropriate genetic counseling. Published 2007 Wiley‐Liss, Inc.

Central nervous system anomalies in Seckel syndrome: Report of a new family and review of the literature

Seckel syndrome (SS) is a rare, heterogeneous form of primordial dwarfism. The clinical delineation of this disorder has been inconsistent, using even Seckels original criteria. As a result, probably fewer than one-third of reported cases are truly affected with SS. Among these, there have been only six familial cases, all of whom were born to normal parents, and in only one case has a detailed description of the central nervous system (CNS) anomalies been given. We describe a family in which three of eight children were affected with SS. CNS anomalies seen in our patients included agenesis of the corpus callosum, a dysgenetic cerebral cortex, a large dorsal cerebral cyst, and pachygyria, suggesting an underlying neuronal migration disorder. The parents are first cousins, representing only the second instance of consanguinity, supporting an autosomal recessive mode of inheritance.

Joubert Syndrome 2 (JBTS2) in Ashkenazi Jews Is Associated with a TMEM216 Mutation

Patients with Joubert syndrome 2 (JBTS2) suffer from a neurological disease manifested by psychomotor retardation, hypotonia, ataxia, nystagmus, and oculomotor apraxia and variably associated with dysmorphism, as well as retinal and renal involvement. Brain MRI results show cerebellar vermis hypoplasia and additional anomalies of the fourth ventricle, corpus callosum, and occipital cortex. The disease has previously been mapped to the centromeric region of chromosome 11. Using homozygosity mapping in 13 patients from eight Ashkenazi Jewish families, we identified a homozygous mutation, R12L, in the TMEM216 gene, in all affected individuals. Thirty individuals heterozygous for the mutation were detected among 2766 anonymous Ashkenazi Jews, indicating a carrier rate of 1:92. Given the small size of the TMEM216 gene relative to other JBTS genes, its sequence analysis is warranted in all JBTS patients, especially those who suffer from associated anomalies.

T‐genes and limb bud development

The T‐box family of transcriptional factors is ancient and highly conserved among most species of animals. Haploinsufficiency of multiple T‐box proteins results in severe human congenital malformation syndromes, involving craniofacial, cardiovascular, and skeletal structures. These genes have major roles in embryogenesis, including the development of the limbs. Formation of the limbs begins with a limb bud and its morphogenesis requires complex epithelial–mesenchymal interactions. Recent studies have shown that T, Tbx2, Tbx3, Tbx4, Tbx5, Tbx15, and Tbx18 are all expressed in the limb buds, and many have developmental functions. The study of these genes is clinically relevant as mutations in several of them cause human congenital malformation syndromes. Furthermore, understanding the function and biology of these genes is important in understanding normal embryogenesis.

Exome Sequence Identifies RIPK4 as the Bartsocas- Papas Syndrome Locus

Pterygium syndromes are complex congenital disorders that encompass several distinct clinical conditions characterized by multiple skin webs affecting the flexural surfaces often accompanied by craniofacial anomalies. In severe forms, such as in the autosomal-recessive Bartsocas-Papas syndrome, early lethality is common, complicating the identification of causative mutations. Using exome sequencing in a consanguineous family, we identified the homozygous mutation c.1127C>A in exon 7 of RIPK4 that resulted in the introduction of the nonsense mutation p.Ser376X into the encoded ankyrin repeat-containing kinase, a protein that is essential for keratinocyte differentiation. Subsequently, we identified a second mutation in exon 2 of RIPK4 (c.242T>A) that resulted in the missense variant p.Ile81Asn in the kinase domain of the protein. We have further demonstrated that RIPK4 is a direct transcriptional target of the protein p63, a master regulator of stratified epithelial development, which acts as a nodal point in the cascade of molecular events that prevent pterygium syndromes.

Genotype and cardiovascular phenotype correlations with TBX1 in 1,022 velo-cardio-facial/DiGeorge/22q11.2 deletion syndrome patients.

Haploinsufficiency of TBX1, encoding a T‐box transcription factor, is largely responsible for the physical malformations in velo‐cardio‐facial /DiGeorge/22q11.2 deletion syndrome (22q11DS) patients. Cardiovascular malformations in these patients are highly variable, raising the question as to whether DNA variations in the TBX1 locus on the remaining allele of 22q11.2 could be responsible. To test this, a large sample size is needed. The TBX1 gene was sequenced in 360 consecutive 22q11DS patients. Rare and common variations were identified. We did not detect enrichment in rare SNP (single nucleotide polymorphism) number in those with or without a congenital heart defect. One exception was that there was increased number of very rare SNPs between those with normal heart anatomy compared to those with right‐sided aortic arch or persistent truncus arteriosus, suggesting potentially protective roles in the SNPs for these phenotype‐enrichment groups. Nine common SNPs (minor allele frequency, MAF > 0.05) were chosen and used to genotype the entire cohort of 1,022 22q11DS subjects. We did not find a correlation between common SNPs or haplotypes and cardiovascular phenotype. This work demonstrates that common DNA variations in TBX1 do not explain variable cardiovascular expression in 22q11DS patients, implicating existence of modifiers in other genes on 22q11.2 or elsewhere in the genome. Hum Mutat 32:1278–1289, 2011. ©2011 Wiley Periodicals, Inc.

Mutations in GJB2, GJB6, and mitochondrial DNA are rare in African American and Caribbean Hispanic individuals with hearing impairment

Autosomal recessive nonsyndromic sensorineural hearing impairment (ARNSHI) comprises 80% of familial hearing loss cases. Approximately half result from mutations in the connexin 26 (Cx26) gene, GJB2, in Caucasian populations. Heterozygous mutations in GJB2 occasionally co‐occur with a deletion of part of GJB6 (connexin 30; Cx30). It is estimated that approximately 1% of deafness is maternally inherited, due to mutations in mitochondrial DNA (mtDNA). Few studies have focused on the frequency of mutations in connexins or mtDNA in African American (AA) and Caribbean Hispanic (CH) admixture populations. In this study, we performed bidirectional sequencing of the GJB2 gene and polymerase chain reaction (PCR) screening for the common GJB6 deletion, as well as PCR/RFLP analysis for three mutations in mtDNA (A1555G, A3243G, A7445G), in 109 predominantly simplex AA and CH individuals. Variations found were a 101T > C (M34T; 1/101 cases), 109G > A (V37I; 1/101), 35delG (mutation; 4/101, ¾ of non‐AA/CH ethnicity), 167delT (mutation; 1/101), 139G > T (mutation; E47X; 1/101 homozygote, consanguineous), −15C > T (1/101), 79G > A (V27I; 9/101), 380G > A (R127H; 4/101; Guyana, India, Pakistan ethnicity), 670A > C (Indeterminate; K224Q; 1/101), 503A > G (novel; K168R; 3/101) and 684C > A (novel; 1/101). All but one of the AA and CH patients had monoallelic variations. There were no hemizygous GJB6 deletions in those with monoallelic GJB2 variations. We also did not identify any patients with the three mutations in mtDNA. Bidirectional sequencing of the GJB2 gene was performed in 187 AA and Hispanic healthy individuals. Our results reveal that GJB2 mutations, GJB6 deletions, and mtDNA mutations may not be significant in these minority admixture populations.

Multicolour FISH and quantitative PCR can detect submicroscopic deletions in holoprosencephaly patients with a normal karyotype

Holoprosencephaly (HPE) is the most common structural malformation of the developing forebrain. At birth, nearly 50% of children with HPE have cytogenetic anomalies. Approximately 20% of infants with normal chromosomes have sequence mutations in one of the four main HPE genes (SHH, ZIC2, SIX3, and TGIF). The other non-syndromic forms of HPE may be due to environmental factors or mutations in other genes, or potentially due to submicroscopic deletions of HPE genes. We used two complementary assays to test for HPE associated submicroscopic deletions. Firstly, we developed a multicolour fluorescent in situ hybridisation (FISH) assay using probes for the four major HPE genes and for two candidate genes (DISP1 and FOXA2). We analysed lymphoblastoid cell lines (LCL) from 103 patients who had CNS findings of HPE, normal karyotypes, and no point mutations, and found seven microdeletions. We subsequently applied quantitative PCR to 424 HPE DNA samples, including the 103 samples studied by FISH: 339 with CNS findings of HPE, and 85 with normal CNS and characteristic HPE facial findings. Microdeletions for either SHH, ZIC2, SIX3, or TGIF were found in 16 of the 339 severe HPE cases (that is, with CNS findings; 4.7%). In contrast, no microdeletion was found in the 85 patients at the mildest end of the HPE spectrum. Based on our data, microdeletion testing should be considered as part of an evaluation of holoprosencephaly, especially in severe HPE cases.

Retinoid signaling in inner ear development: A “Goldilocks” phenomenon

Retinoic acid (RA) is a biologically active derivative of vitamin A that is indispensable for inner ear development. The normal function of RA is achieved only at optimal homeostatic concentrations, with an excess or deficiency in RA leading to inner ear dysmorphogenesis. We present an overview of the role of RA in the developing mammalian inner ear, discussing both how and when RA may act to critically control a program of inner ear development. Molecular mechanisms of otic teratogenicity involving two members of the fibroblast growth factor family, FGF3 and FGF10, and their downstream targets, Dlx5 and Dlx6, are examined under conditions of both RA excess and deficiency. We term the effect of too little or too much RA on FGF/Dlx signaling a Goldilocks phenomenon. We demonstrate that in each case (RA excess, RA deficiency), RA can directly affect FGF3/FGF10 signaling within the otic epithelium, leading to downregulated expression of these essential signaling molecules, which in turn, leads to diminution in Dlx5/Dlx6 expression. Non‐cell autonomous affects of the otic epithelium subsequently occur, altering transforming growth factor‐beta (TGFβ) expression in the neighboring periotic mesenchyme and serving as a putative explanation for RA‐mediated otic capsule defects. We conclude that RA coordinates inner ear morphogenesis by controlling an FGF/Dlx signaling cascade, whose perturbation by deviations in local retinoid concentrations can lead to inner ear dysmorphogenesis.