We observed two unrelated consanguineous families with malformation syndromes sharing anophthalmia and distinct eyebrows as common signs, but differing for alveolar capillary dysplasia or complex congenital heart defect in one and diaphragmatic hernia in the other family. Homozygosity mapping revealed linkage to a common locus on chromosome 15, and pathogenic homozygous mutations were identified in STRA6, a member of a large group of “stimulated by retinoic acid” genes encoding novel transmembrane proteins, transcription factors, and secreted signaling molecules or proteins of largely unknown function. Subsequently, homozygous STRA6 mutations were also demonstrated in 3 of 13 patients chosen on the basis of significant phenotypic overlap to the original cases. While a homozygous deletion generating a premature stop codon (p.G50AfsX22) led to absence of the immunoreactive protein in patient’s fibroblast culture, structural analysis of three missense mutations (P90L, P293L, and T321P) suggested significant effects on the geometry of the loops connecting the transmembrane helices of STRA6. Two further variations in the C-terminus (T644M and R655C) alter specific functional sites, an SH2-binding motif and a phosphorylation site, respectively. STRA6 mutations thus define a pleiotropic malformation syndrome representing the first human phenotype associated with mutations in a gene from the “STRA” group. We observed two unrelated consanguineous families with malformation syndromes sharing anophthalmia and distinct eyebrows as common signs, but differing for alveolar capillary dysplasia or complex congenital heart defect in one and diaphragmatic hernia in the other family. Homozygosity mapping revealed linkage to a common locus on chromosome 15, and pathogenic homozygous mutations were identified in STRA6, a member of a large group of “stimulated by retinoic acid” genes encoding novel transmembrane proteins, transcription factors, and secreted signaling molecules or proteins of largely unknown function. Subsequently, homozygous STRA6 mutations were also demonstrated in 3 of 13 patients chosen on the basis of significant phenotypic overlap to the original cases. While a homozygous deletion generating a premature stop codon (p.G50AfsX22) led to absence of the immunoreactive protein in patient’s fibroblast culture, structural analysis of three missense mutations (P90L, P293L, and T321P) suggested significant effects on the geometry of the loops connecting the transmembrane helices of STRA6. Two further variations in the C-terminus (T644M and R655C) alter specific functional sites, an SH2-binding motif and a phosphorylation site, respectively. STRA6 mutations thus define a pleiotropic malformation syndrome representing the first human phenotype associated with mutations in a gene from the “STRA” group. Clinical anophthalmia (AO) is the complete absence of the eye and may be the most severe end of a clinical spectrum of ocular malformations including microphthalmia (MO), which is a small eye usually defined in terms of corneal diameter or axial length.1Morrison D FitzPatrick D Hanson I Williamson K van Heyningen V Fleck B Jones I Chalmers J Campbell H National study of microphthalmia, anophthalmia, and coloboma (MAC) in Scotland: investigation of genetic aetiology.J Med Genet. 2002; 39: 16-22Crossref PubMed Scopus (191) Google Scholar Estimates of the birth prevalence of anophthalmia and microphthalmia from well-maintained population-based registers are 14 and 3 per 100,000 births, respectively.1Morrison D FitzPatrick D Hanson I Williamson K van Heyningen V Fleck B Jones I Chalmers J Campbell H National study of microphthalmia, anophthalmia, and coloboma (MAC) in Scotland: investigation of genetic aetiology.J Med Genet. 2002; 39: 16-22Crossref PubMed Scopus (191) Google Scholar Autosomal recessive origin is likely in ∼10% of cases.2Vogt G Puho E Czeizel AE A population-based case-control study of isolated anophthalmia and microphthalmia.Eur J Epidemiol. 2005; 20: 939-946Crossref PubMed Scopus (9) Google ScholarCHX10 mutations have been shown to underlie autosomal recessive isolated clinical anophthalmia in two families3Bar-Yosef U Abuelaish I Harel T Hendler N Ofir R Birk OS CHX10 mutations cause non-syndromic microphthalmia/ anophthalmia in Arab and Jewish kindreds.Hum Genet. 2004; 115: 302-309Crossref PubMed Scopus (68) Google Scholar and microphthalmia with cataract and abnormalities of the iris,4Ferda Percin E Ploder LA Yu JJ Arici K Horsford DJ Rutherford A Bapat B Cox DW Duncan AM Kalnins VI et al.Human microphthalmia associated with mutations in the retinal homeobox gene CHX10.Nat Genet. 2000; 25: 397-401Crossref PubMed Scopus (243) Google Scholar whereas syndromic autosomal dominant anophthalmia has been associated with mutations in SOX2.5Fantes J Ragge NK Lynch SA McGill NI Collin JR Howard-Peebles PN Hayward C Vivian AJ Williamson K van Heyningen V et al.Mutations in SOX2 cause anophthalmia.Nat Genet. 2003; 33: 461-463Crossref PubMed Scopus (430) Google Scholar According to a national study on microphthalmia, anophthalmia, and coloboma, 33% of cases had one or more associated major malformations, and 21% had learning disabilities.1Morrison D FitzPatrick D Hanson I Williamson K van Heyningen V Fleck B Jones I Chalmers J Campbell H National study of microphthalmia, anophthalmia, and coloboma (MAC) in Scotland: investigation of genetic aetiology.J Med Genet. 2002; 39: 16-22Crossref PubMed Scopus (191) Google Scholar As only a few of the latter syndromal cases had features of recognized entities, it was assumed that several new syndrome diagnoses are yet to be delineated in this group. To increase the understanding of syndromic anophthalmia, we performed positional cloning in two unrelated consanguineous families with new, apparently not yet reported conditions, including clinical anophthalmia and variable malformations of the lung, the heart, and the diaphragm, as well as mental retardation. This work was performed as part of our research study addressing the genetics of mental retardation, which was approved by the research ethics committee of the University of Erlangen-Nuremberg. The proband in family 1 (IV:2 in fig. 1A) was a female infant born at 33 wk of gestation following a pregnancy during which bilateral anophthalmia had been diagnosed by ultrasound scan at 16 wk of gestation. She had normal intrauterine growth, with a birth length of 49 cm (97th percentile), weight 2,035 g (50th percentile), and head circumference 32 cm (75th percentile). In the perinatal period, bilateral clinical anophthalmia was confirmed. Additional malformations were noted at that time: right-sided pelvic kidney, circulatory nonrelevant pulmonic valve stenosis, and persistent ductus arteriosus, which was surgically closed at the age of 3 wk. When assessed at the age of 2 mo, growth was normal, with the crown-to-heel length (50 cm) and weight (3,200 g) at the age of 2 mo corresponding to the 10th–25th percentile for prematurely born girls, whereas head circumference (35 cm) corresponded to the 25th–50th percentile. She had mild facial dysmorphism, with marked blepharophimosis with an unusual trichoglyphic pattern of the eyebrows, which were broad, flaring, and only upward growing (fig. 2E). She had a broad nasal bridge, micrognathia, and large, low-set ears (fig. 2A and 2B). Cerebral magnetic resonance imaging (MRI) showed no abnormality of brain structure and showed visible optic nerves and chiasm. Mechanical ventilatory support was required from birth, because of persistent respiratory insufficiency. Chest CT at the age of 6 wk showed no evidence of pulmonary malformations, lymphangiectasia, or interstitial lung disease. Open lung biopsy performed at the age of 2.5 mo revealed a reduced number of alveolar units and pulmonary capillary vessels with thickening of the interalveolar septa, as well as medial thickening of small pulmonary arteries with muscularization, which are the key features of alveolar capillary dysplasia (MIM 235680) (fig. 2G) without misalignment of lung vessels. She did not show any psychomotor development and, despite high-dose steroid treatment, she was extubated for only 13 d before dying at the age of 6 mo from respiratory insufficiency. The family history was significant. The parents are first cousins of Turkish origin. The paternal uncle of the proband, who was also married to his half-cousin, had a daughter with bilateral anophthalmia, who died at the age of 2 d from a complex cyanotic congenital heart defect with atresia of the pulmonary artery and single ventricle (IV:4 in fig. 1A). She had normal intrauterine growth, with a birth length of 51 cm (25th–50th percentile), weight 3,240 g (25th–50th percentile), and head circumference 36 cm (75th percentile). Karyotype, metabolic screen, and cerebral and renal ultrasound all showed normal results. Parents consented to postmortem examination of thoracic organs, which confirmed the single ventricle with atresia of the pulmonary artery.Figure 2A–D, Frontal and lateral views of patient IV:2 of family 1 at age 6 mo (A and B) and patient IV:1 of family 2 at age 13 years (C and D). Note similar mild dysmorphism with broad, flaring, and only upward-growing eyebrows; broad nasal bridge; large, low-set ears; and receding chin. E and F, Close-up of right eyebrows of IV:2 of family 1 (E) and IV:1 of family 2 (F). G, Hematoxylin-eosin staining of lung biopsy, showing deficiency in the number of alveolar units and pulmonary capillary vessels with thickening of the interalveolar septa.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The proband in family 2 (IV:1 in fig. 1B) was a 14-year-old boy with bilateral clinical anophthalmia, diaphragmatic hernia, and profound mental retardation, with a performance IQ estimated to be <20. He was the eldest son of a healthy consanguineous couple of Turkish origin. When assessed at the age of 13 years 3 mo, he had severe short stature (height 123 cm [−4.47 SD]; weight 21 kg [BMI −2.95]) with relative preservation of head growth (occipitofrontal circumference 51 cm [3rd percentile]). He had no speech and had no obvious receptive language skills. Although he used a wheelchair, he was able to take a few steps when supported. He had both an atrial and a ventricular septal defect, which did not require any therapy. Cerebral MRI performed at the age of 4 years showed a structurally normal brain, apart from absent optic nerves. He had mild facial dysmorphism with severe blepharophimosis and an unusual trichoglyphic pattern of both eyebrows similar to that seen in family 1 (fig. 2F). He had a broad nasal bridge, micrognathia, and large, low-set ears (fig. 2C and 2D). He had a healthy brother and sister. His mother had had one termination of pregnancy because of perceived high risk and another pregnancy that was terminated at 23 wk gestation after a diagnosis of bilateral anophthalmia and severe diaphragmatic hernia on antenatal ultrasound scan (IV:3). The fetus showed mild facial dysmorphism similar to that of the probands in this family and family 1. Parents did not consent to postmortem examination but agreed to skin biopsy for fibroblast culture. To identify the underlying disease genes, a genomewide linkage scan was performed using the Affymetrix GeneChip Human Mapping 10K SNP array Xba142 (version 2.0) and both affected and unaffected individuals from both families. The sex of each sample was verified by counting heterozygous SNPs on the X chromosome. Relationship errors were evaluated with the help of the program Graphical Relationship Representation.6Abecasis GR Cherny SS Cookson WO Cardon LR GRR: graphical representation of relationship errors.Bioinformatics. 2001; 17: 742-743Crossref PubMed Scopus (345) Google Scholar The program PedCheck was applied to detect Mendelian errors,7O’Connell JR Weeks DE PedCheck: a program for identification of genotype incompatibilities in linkage analysis.Am J Hum Genet. 1998; 63: 259-266Abstract Full Text Full Text PDF PubMed Scopus (1822) Google Scholar and data for SNPs with such errors were removed from the data set. Non-Mendelian errors were identified by use of the program MERLIN,8Abecasis GR Cherny SS Cookson WO Cardon LR Merlin—rapid analysis of dense genetic maps using sparse gene flow trees.Nat Genet. 2002; 30: 97-101Crossref PubMed Scopus (2780) Google Scholar and unlikely genotypes for related samples were deleted. LOD score calculations were performed using the Allegro program,9Gudbjartsson DF Jonasson K Frigge ML Kong A Allegro, a new computer program for multipoint linkage analysis.Nat Genet. 2000; 25: 12-13Crossref PubMed Scopus (674) Google Scholar under the assumption of autosomal recessive inheritance with full penetrance. The parametric analysis unveiled the expected maximum multipoint LOD score of 2.9 on chromosome 15 (q23-25.1) in family 1 for a region of ∼15 cM (fig. 3A) and multiple possible loci in family 2, including the same locus on chromosome 15 with the expected maximum LOD score of 1.9 (fig. 3B). Parametric and nonparametric linkage analysis of both families together revealed a single maximum LOD score of 4.8 for the region 15q23-25.1, which was also achieved when allowing for locus heterogeneity (HLOD) (fig. 3C). Haplotypes were reconstructed with ALLEGRO and were presented graphically with HaploPainter.10Thiele H Nurnberg P HaploPainter: a tool for drawing pedigrees with complex haplotypes.Bioinformatics. 2005; 21: 1730-1732Crossref PubMed Scopus (231) Google Scholar This latter program also reveals informative SNP markers as points of recombination between parental haplotypes. All data handling was performed using the graphical user interface ALOHOMORA.11Ruschendorf F Nurnberg P ALOHOMORA: a tool for linkage analysis using 10K SNP array data.Bioinformatics. 2005; 21: 2123-2125Crossref PubMed Scopus (152) Google Scholar Haplotype reconstruction showed homozygosity for two different alleles at the same 12-Mb region between markers SNP_A-1511966 (rs1822829) and SNP_A-1509050 (rs1077965) in affected children from both families (fig. 4).Figure 4Haplotype analyses on chromosome 15q23-25.1, showing homozygous markers linked to the disease locus in family 1 (A) and family 2 (B). According to the NCBI human genome overview page, build 35.1, the flanking markers SNP_A-1511966 (rs1822829) and SNP_A-1509050 (rs1077965) span a region of ∼12 Mb between 65.21 and 77.85 Mb from pter.View Large Image Figure ViewerDownload Hi-res image Download (PPT) This critical region contained >280 known and predicted genes annotated in the UCSC database. After evaluation of the available data on developmental expression and function of each of the genes, we selected two for mutational analysis. UACA (encoding uveal autoantigen with coiled-coil domains and ankyrin repeats protein) was selected because it plays an important role in the regulation of stress-induced apoptosis and because it is expressed in heart and in choroid, retina, and eye muscles, among other tissues, according to the SwissProt protein database. STRA6 (FLJ12541; Stra for “stimulated by retinoic acid”) was chosen because of its involvement in the retinoic acid pathway and its therefore likely role in morphogenesis, as well as reported expression in mouse tissues corresponding to affected organs in our patients. Sequencing of UACA revealed no mutation in the probands of family 1 (IV:2) and 2 (IV:1). Sequence analysis of all 20 exons (fig. 5A) and intronic flanking regions of STRA6 revealed single-nucleotide variants in both families. A homozygous missense mutation in exon 12, c.878C→T (P293L), was found in the affected child IV:2 of family 1 (fig. 1C). A homozygous frameshift mutation leading to a premature stop codon (c.145_147delC; p.G50AfsX22) in exon 4 was demonstrated in both affected children of family 2 (fig. 1D). Both mutations were shown to cosegregate with the disease phenotype in the respective families (fig. 1A and 1B). After the identification of STRA6 mutations in these families, the mutational analysis was extended using a cohort of 13 unrelated white patients selected on the basis of having a severe eye malformation and malformations of the diaphragm or one of the latter associated with malformations of the lungs or heart (table 1). This analysis led to the identification of four further homozygous amino acid changes (P90L, T321P, T644M, and R655C) in STRA6 in three patients (tables 1 and 2 and fig. 5A). Parental heterozygosity for the respective mutation was proved in families for which parental samples were available (MWS1-EE and MWS4-BE). Patient MWS6-BK carries two of these missense mutations (P90L and T321P), both homozygously, but no other family members were available for further analysis. None of the mutations were found in a panel of 190 healthy, adult white control individuals. Further support for the pathogenicity of the missense mutations was provided by the demonstration that each of the substituted amino acids was evolutionarily conserved, which was done using multiple sequence alignments by ClustalW (fig. 5B). RT-PCR analysis of RNA isolated from cultured fibroblasts of the affected aborted fetus (IV:3) from family 2, which harbored the premature stop mutation, showed detectable levels of the mutated STRA6 transcript with and without puromycin treatment (fig. 6A). Western blot analysis of protein extracted from these fibroblasts was performed using a rabbit polyclonal antibody raised to the C-terminal region of STRA612Sapin V Bouillet P Oulad-Abdelghani M Dastugue B Chambon P Dolle P Differential expression of retinoic acid-inducible (Stra) genes during mouse placentation.Mech Dev. 2000; 92: 295-299Crossref PubMed Scopus (42) Google Scholar (kindly supplied by Pierre Chambon). This showed absence of immunoreactive protein in the patient’s fibroblasts (fig. 6B). RT-PCR on normal adult human mRNAs from different tissues confirmed the broad expression pattern previously found in mice (fig. 7A). Detailed analysis of distinct parts of an adult human eye revealed expression in sclera, retina, retinal pigment epithelium, and trabecular mashwork but not in choroid and iris (fig. 7B).Table 1Overview of Phenotype in Patients Investigated for STRA6 MutationsPatientMutationParental ConsanguinityEyeLungDiaphragmHeartPalateKidneyUterusAge at DeathOtherFam1-IV:2P293L+b AOACD−PSt, PDA−Pelvic−6 moPTB 33 weeks, DDFam1-IV:4NA+b AO−−CHD,PA−−−2 dFam2-IV:1p.G50AfsX22+b AO−CDHASD, VSD−−−Alive at age 14 years, profound MR, SOSFam2-IV:3p.G50AfsX22+b AO−CDH−−−−TOPMWS1-EER655C+b AOHypoDE3 moInguinal hernia, severe hypotonia, failure to thrive BrotherNA+b AO−−TAC-IV, RAA, PDA, PA−−−22 moSOSMWS4-BET644M−b AOHypoCDHb hydronephrosisAlive at age 3 mo BrotherNA−?Hypo, unilo−TOF, PDA−Horseshoe−1 dUndescended testes, hypoplastic renal arteries SisterNA−b AOHypo, unilo−PDA, CoA−−Dysplasia1 dMWS6-BKP90L, T321P+b AOHypoCDH, DEPDAHypoBicornuate1 dPTB 36 wk, Meckel diverticleMWS2-FA−+b Col−CDH−−−−Skin patches, brittle hairMWS3-KH−−b MO−CDH−−−−MO: extremeMWS5-LR−−Col−CDH−−−−RHP006.070−−b MO−b DE−−−−MO: extreme, MRGM23728−−b MOHypo, uniloDE, hypoHypo Pa, CoADysplasticNeonatalAbnormal cornea and irisAvdW22260 -Twin1−−−HypoCDH−−−−1 dPTB (28 wk) Twin 2−−−HypoCDH−CP−−1 dPTB (28 wk)AS20861-FF264−−ri MO−CDH−−−−MO: max. diameter 9 mm at age 13 mo with internal, dense calcification within the globe, and a larger, inferiorly located benign cyst measuring 2.5 cm; DD (11 mo level at age 13 mo)CD50396−−b AOHypoDEVSDCP−Hypo, bicornuate1 dMatthew-Woods syndrome, hypoplastic spleen, hypoplastic alae nasiPM22479−+−−CDH−−−−NeonatalSuspected Donnai-Barrow syndrome (MIM 222448), large omphalocele, hypoplasia of corpus callosum, enlarged ventricles, extreme hypertelomerism BrotherNA+−−CDHASDb CLP−−NeonatalSuspected Donnai-Barrow syndrome (MIM 222448), hypoplasia of corpus callosum, enlarged ventricles, extreme hypertelomerismPB-E03_053−−b MO−CDH−−−−−MO: severe, b inguinal hernia, sparse hair, brachycephaly, MR, spasticity, alive at 10 yearsNote.—ACD = alveolar capillary dysplasia; AO = anophthalmia; ASD = artrial septal defect; b = bilateral; CDH = congenital diaphragmatic hernia; CHD = congenital heart defect; C(L)P = cleft (lip) palate; CoA = coarctation of aorta; Col = coloboma; DD = developmental delay; DE = diaphragmatic eventration; Hypo = hypoplastic; MO = microphthalmia; MR = mental retardation; NA = not analyzed; Pa = pulmonary artery; PA = atresia of pulmonary artery; PDA = persistent ductus arteriosus; PSt = pulmonic valve stenosis; PTB = preterm birth; RAA = right aortic arch; ri = right sided; SOS = postnatal shortness of stature; TAC = truncus arteriosus communis; TOF = tetralogy of Fallot; TOP = termination of pregnancy; unilob = unilobular lung; VSD = ventricular septal defect. Open table in a new tab Table 2Overview of STRA6 MutationsAlterationaAll mutations were homozygous.PatientExonGenomicProteinFam1-IV:212c.878C→TP293LFam2-IV:14c.145-147delCp.G50AfsX22Fam2-IV:34c.145-147delCp.G50AfsX22MWS1-EE20c.1963C→TR655CMWS4-BE20c.1931C→TT644MMWS6-BK6, 13c.269C→T, c.961A→CP90L, T321Pa All mutations were homozygous. Open table in a new tab Figure 6Characterization of mutations. A, STRA6 RT-PCR on cultured fibroblast cells from the affected fetus (IV:3, family 2) and from a healthy individual (C) grown in the absence and the presence of puromycin (p−/p+) as inhibitor of translation, respectively, showing no evidence for early nonsense-mediated mRNA decay; (+) positive control, (−) negative control. B, Western blot analysis of cultured fibroblast protein cell extract showing a STRA6 protein band in two human healthy control lanes (indicated as “WT”) but not in the two lanes with the homozygous mutant c.145_147delC (from IV:3, family 2). C, Model showing the effect of the P293L mutation (right) on the STRA6 structure in comparison with the wild type (left). The membrane is indicated by a dotted line. The transmembrane helix (A300-V319) that is proximal to the site of the mutation is shown in ribbon presentation, and the two adjacent helices are indicated by cylinders. In the wild type, residue P293 represents the N-terminal cap of a helix starting at L294, whereas the L293 present in the mutant allows an N-terminal extension of the helix, now starting at H291 and changing the topology of the respective loop. Therefore, the P293L mutation is predicted to cause an extension of the helix by three residues, thus affecting the structure and orientation of the respective loop. Moreover, it is important to note that V319 is also the N-terminal residue of an extremely short loop (maximum predicted length between aa 319 and aa 326), in which a second mutation (T321P) has been identified.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Expression pattern of STRA6 determined by RT-PCR in normal adult human (A) and eye (B) tissues. RPE = Retinal pigment epithelium; TM = Trabecular meshwork.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Note.— ACD = alveolar capillary dysplasia; AO = anophthalmia; ASD = artrial septal defect; b = bilateral; CDH = congenital diaphragmatic hernia; CHD = congenital heart defect; C(L)P = cleft (lip) palate; CoA = coarctation of aorta; Col = coloboma; DD = developmental delay; DE = diaphragmatic eventration; Hypo = hypoplastic; MO = microphthalmia; MR = mental retardation; NA = not analyzed; Pa = pulmonary artery; PA = atresia of pulmonary artery; PDA = persistent ductus arteriosus; PSt = pulmonic valve stenosis; PTB = preterm birth; RAA = right aortic arch; ri = right sided; SOS = postnatal shortness of stature; TAC = truncus arteriosus communis; TOF = tetralogy of Fallot; TOP = termination of pregnancy; unilob = unilobular lung; VSD = ventricular septal defect. Both the function and the tertiary structure of STRA6 peptide are unknown. To explain the potential effects of the missense mutations, we performed secondary structure analysis by use of three different prediction programs (TMHMM,13Krogh A Larsson B von Heijne G Sonnhammer EL Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes.J Mol Biol. 2001; 305: 567-580Crossref PubMed Scopus (9122) Google ScholarTMpred,14Reddy EP Korapati A Chaturvedi P Rane S IL-3 signaling and the role of Src kinases, JAKs and STATs: a covert liaison unveiled.Oncogene. 2000; 19: 2532-2547Crossref PubMed Scopus (191) Google Scholar and TopPred15Claros MG von Heijne G TopPred II: an improved software for membrane protein structure predictions.Comput Appl Biosci. 1994; 10: 685-686PubMed Google Scholar), which suggested that STRA6 has between 8 and 12 transmembrane helices. Accordingly, three missense mutations (P90L, P293L, and T321P) are predicted to be located in loops connecting these transmembrane helices, whereas two missense mutations (T644M and R655C) are located in the evolutionary conserved C-terminal region of the protein. Analysis of the secondary structure outside the transmembrane segments was performed using a consensus secondary prediction from the [email protected] server.16Combet C Blanchet C Geourjon C Deleage G [email protected]: network protein sequence analysis.Trends Biochem Sci. 2000; 25: 147-150Abstract Full Text Full Text PDF PubMed Scopus (1435) Google Scholar This approach suggested that the mutations P293L and P90L increase the extent of helical structures in STRA6, resulting in an extension of the transmembrane helix (P293L) (fig. 6C) and the formation of a novel helix within a loop (P90L). As a consequence, the topology of the loops is altered, and numerous conserved residues are brought into a different orientation with respect to the membrane. Amino acid T321 was also predicted to be located in an extremely short loop (maximum length comprising residues 319–326) connecting two transmembrane helices. As a consequence of the T321P mutation, a rigid diproline-motif (P320–P321) is generated that is probably incompatible with the sterically demanding topology of this tight loop (fig. 6C). Thus, mutants P90L and T321P, observed homozygously in the same patient, are both expected to have significant effects on the secondary and tertiary structure of the loops in STRA6. In contrast, mutations T644M and R655C are predicted by the ELM program17Puntervoll P Linding R Gemund C Chabanis-Davidson S Mattingsdal M Cameron S Martin DM Ausiello G Brannetti B Costantini A et al.ELM server: a new resource for investigating short functional sites in modular eukaryotic proteins.Nucleic Acids Res. 2003; 31: 3625-3630Crossref PubMed Scopus (519) Google Scholar to impair functional sites at the C-terminal region. T644M alters the STAT5 Src Homology 2 (SH2) domain binding motif, YTLL, which triggers the JAK2/STAT5 signaling cascade. STAT5 and related members of the STAT family are activated in different tissues by means of a series of ligands and are involved in interferon signaling, development of the mammary gland, response to growth hormone, and embryogenesis.18Calo V Migliavacca M Bazan V Macaluso M Buscemi M Gebbia N Russo A STAT proteins: from normal control of cellular events to tumorigenesis.J Cell Physiol. 2003; 197: 157-168Crossref PubMed Scopus (525) Google Scholar R655C alters the invariant arginine of the R-X-[ST] consensus sequence representing a phosphorylation site of proteinkinase A19Shabb JB Physiological substrates of cAMP-dependent protein kinase.Chem Rev. 2001; 101: 2381-2411Crossref PubMed Scopus (282) Google Scholar and therefore probably alters the successive signal transduction pathways. The phenotype of the three patients detected on the follow-up mutation analysis shows remarkable overlap with that of the initial cases, with bilateral clinical anophthalmia and normal birth measurements as consistent features. The healthy parents of patient MWS1-EE are distantly related through a common great-great-grandparent. After a pregnancy remarkable for polyhydramnion, MWS1-EE was born at term with normal measurements (weight 3,130 g [10th–25th percentile], length 50 cm [10th–25th percentile], and head circumference 35 cm [25th–50th percentile]). Because of respiratory insufficiency, ventilatory support was needed for 4 d. The boy showed bilateral anophthalmia, left-sided diaphragmatic eventration, and right-sided inguinal hernia. He had severe hypotonia, poor feeding, and almost no weight gain until he died at the age of 3 mo. No postmortem examination was performed. The older brother of MWS1-EE also showed bilateral anophthalmia, with only remains of nervi optici detected at autopsy. In addition, he had truncus arteriosus communis type IV with right-sided aorta, lack of pulmonary arteries, and lung supply by bronchial arteries. He died from a thrombosis of the bronchial arterial branches at the age of 22 mo. Although his birth measurements at 36 wk gestation were normal (weight 2,300 g [10th percentile], length 46 cm [10th–25th percentile], and head circumference 33 cm [25th–50th percentile]), he had short stature at autopsy (length 78 cm [−3.26 SD] and head circumference 48 cm [25th–50th percentile]). He was not able to walk bu
