Sarah Dyack

Mutations in mitochondrial carrier family gene SLC25A38 cause nonsyndromic autosomal recessive congenital sideroblastic anemia

The sideroblastic anemias are a heterogeneous group of congenital and acquired hematological disorders whose morphological hallmark is the presence of ringed sideroblasts—bone marrow erythroid precursors containing pathologic iron deposits within mitochondria. Here, by positional cloning, we define a previously unknown form of autosomal recessive nonsyndromic congenital sideroblastic anemia, associated with mutations in the gene encoding the erythroid specific mitochondrial carrier family protein SLC25A38, and demonstrate that SLC25A38 is important for the biosynthesis of heme in eukaryotes.

Recurrent Gain-of-Function Mutation in PRKG1 Causes Thoracic Aortic Aneurysms and Acute Aortic Dissections

Gene mutations that lead to decreased contraction of vascular smooth-muscle cells (SMCs) can cause inherited thoracic aortic aneurysms and dissections. Exome sequencing of distant relatives affected by thoracic aortic disease and subsequent Sanger sequencing of additional probands with familial thoracic aortic disease identified the same rare variant, PRKG1 c.530G>A (p.Arg177Gln), in four families. This mutation segregated with aortic disease in these families with a combined two-point LOD score of 7.88. The majority of affected individuals presented with acute aortic dissections (63%) at relatively young ages (mean 31 years, range 17-51 years). PRKG1 encodes type I cGMP-dependent protein kinase (PKG-1), which is activated upon binding of cGMP and controls SMC relaxation. Although the p.Arg177Gln alteration disrupts binding to the high-affinity cGMP binding site within the regulatory domain, the altered PKG-1 is constitutively active even in the absence of cGMP. The increased PKG-1 activity leads to decreased phosphorylation of the myosin regulatory light chain in fibroblasts and is predicted to cause decreased contraction of vascular SMCs. Thus, identification of a gain-of-function mutation in PRKG1 as a cause of thoracic aortic disease provides further evidence that proper SMC contractile function is critical for maintaining the integrity of the thoracic aorta throughout a lifetime.

Heritable skewed X-chromosome inactivation leads to haemophilia A expression in heterozygous females

Factor VIII gene, F8, mutations cause haemophilia A (HA), an X-linked recessive disorder. Expression in heterozygous females has been ascribed to skewed X-chromosome inactivation (XCI). To investigate the cause of HA in three heterozygous females within an Atlantic Canadian kindred, the proband (severely affected girl, FVIII activity: 2%) and 17 relatives across three generations were studied. F8 genotype, FVIII activity, XCI ratio (XCIR) (paternal active X: maternal active X), karyotype, submegabase resolution tiling set array competitive genome hybridization (competitive genomic hybridization (SMRT)), and microsatellite analyses were utilized. A positive linear relationship between FVIII activity and percentage-activated normal X-chromosome was found in HA heterozygous females (R2=0.87). All affected, but no unaffected females, had an XCIR skewed toward activation of the mutant X-chromosome (proband 92:8, SD 2). Unexpectedly, high numbers of females have dramatically skewed XCIRs (>80:20 or <20:80) (P<0.05). The distribution of XCIR frequencies within this family was significantly different than predicted by normal population data or models of random XCI (P<0.025), with more females having higher degrees of skewing. Known causes of skewing, such as chromosomal abnormalities, selection against deleterious alleles, and X-inactive-specific transcript mutations, are not consistent with our results. This study shows that FVIII activity in HA heterozygous females can be directly related to XCI skewing, and that low FVIII activity in females in this family is due to unfavourable XCI skewing. Further, the findings suggest that these XCI ratios are genetically influenced, consistent with a novel heritable human X controlling element (XCE) functioning similarly to the mouse Xce.

Urinary biomarker investigation in children with Fabry disease using tandem mass spectrometry.

BACKGROUND Fabry disease is an X-linked lysosomal storage disorder affecting both males and females with tremendous genotypic/phenotypic variability. Concentrations of globotriaosylceramide (Gb3), globotriaosylsphingosine (lyso-Gb3)/related analogues were investigated in pediatric and adult Fabry cohorts. The aims of this study were to transfer and validate an HPLC-MS/MS methodology on a UPLC-MS/MS new generation platform, using an HPLC column, for urine analysis of treated and untreated pediatric and adult Fabry patients, to establish correlations between the excretion of Fabry biomarkers with gender, treatment, types of mutations, and to evaluate the biomarker reliability for early detection of pediatric Fabry patients. METHOD A UPLC-MS/MS was used for biomarker analysis. RESULTS Reference values are presented for all biomarkers. Results show that gender strongly influences the excretion of each biomarker in the pediatric Fabry cohort, with females having lower urinary levels of all biomarkers. Urinary distribution of lyso-Gb3/related analogues in treated Fabry males was similar to the untreated and treated Fabry female groups in both children and adult cohorts. Children with the late-onset p.N215S mutation had normal urinary levels of Gb3, and lyso-Gb3 but abnormal levels of related analogues. CONCLUSIONS In this study, Fabry males and most Fabry females would have been diagnosed using the urinary lyso-Gb3/related analogue profile.

Germline mutations in MAP3K6 are associated with familial gastric cancer.

Gastric cancer is among the leading causes of cancer-related deaths worldwide. While heritable forms of gastric cancer are relatively rare, identifying the genes responsible for such cases can inform diagnosis and treatment for both hereditary and sporadic cases of gastric cancer. Mutations in the E-cadherin gene, CDH1, account for 40% of the most common form of familial gastric cancer (FGC), hereditary diffuse gastric cancer (HDGC). The genes responsible for the remaining forms of FGC are currently unknown. Here we examined a large family from Maritime Canada with FGC without CDH1 mutations, and identified a germline coding variant (p.P946L) in mitogen-activated protein kinase kinase kinase 6 (MAP3K6). Based on conservation, predicted pathogenicity and a known role of the gene in cancer predisposition, MAP3K6 was considered a strong candidate and was investigated further. Screening of an additional 115 unrelated individuals with non-CDH1 FGC identified the p.P946L MAP3K6 variant, as well as four additional coding variants in MAP3K6 (p.F849Sfs*142, p.P958T, p.D200Y and p.V207G). A somatic second-hit variant (p.H506Y) was present in DNA obtained from one of the tumor specimens, and evidence of DNA hypermethylation within the MAP3K6 gene was observed in DNA from the tumor of another affected individual. These findings, together with previous evidence from mouse models that MAP3K6 acts as a tumor suppressor, and studies showing the presence of somatic mutations in MAP3K6 in non-hereditary gastric cancers and gastric cancer cell lines, point towards MAP3K6 variants as a predisposing factor for FGC.

Deletions in 16q24.2 are associated with autism spectrum disorder, intellectual disability and congenital renal malformation

Background The contribution of copy-number variation (CNV) to disease has been highlighted with the widespread adoption of array-based comparative genomic hybridisation (aCGH) and microarray technology. Contiguous gene deletions involving ANKRD11 in 16q24.3 are associated with autism spectrum disorder (ASD) and intellectual disability (ID), while 16q24.1 deletions affecting FOXF1 are associated with congenital renal malformations, alveolar capillary dysplasia, and various other abnormalities. The disease associations of deletions in the intervening region, 16q24.2, have only been defined to a limited extent. Aim To determine whether deletions affecting 16q24.2 are correlated with congenital anomalies. Methods 35 individuals, each having a deletion in 16q24.2, were characterised clinically and by aCGH and/or SNP-genotyping microarray. Results Several of the 35 16q24.2 deletions identified here closely abut or overlap the coding regions of FOXF1 and ANKRD11, two genes that have been previously associated with the disease. 25 patients were reported to have ASD/ID, and three were found to have bilateral hydronephrosis. 14 of the deletions associated with ASD/ID overlap the coding regions of FBXO31 and MAP1LC3B. These same genes and two others, C16orf95 and ZCCHC14, are also included in the area of minimal overlap of the three deletions associated with hydronephrosis. Conclusions Our data highlight 16q24.2 as a region of interest for ASD, ID and congenital renal malformations. These conditions are associated, albeit without complete penetrance, with deletions affecting C16orf95, ZCCHC14, MAP1LC3B and FBXO31. The function of each gene in development and disease warrants further investigation.

Congenital mydriasis, patent ductus arteriosus, and congenital cystic lung disease: New syndromic spectrum?

Congenital mydriasis is an extremely rare ophthalmic condition involving aplasia of the iris sphincter muscles and hypofunction or hypoplasia of the dilator muscles resulting in fixed dilated pupils, often bilateral [White and Fulton, 1937; Caccamise and Townes, 1976; Richardson and Schulenburg, 1992; Buys et al., 1993; Graf and Jungherr, 2002]. Some cases are associated with developmental delay and White’s cases in monozygotic female twins suggest inheritance to be either autosomal dominant or X-linked dominant with limited viability inmales. Buys et al. [1993] initially reported an association between congenital mydriasis and patent ductus arteriosus in a boy and Graf and Jungherr [2002] later described the defect in two patients, suggesting a causal relationship in the pathogenesis of the two defects. We describe another patient with congenital mydriasis and patent ductus arteriosus, but with additional findings of coarctation of the aorta, congenital cystic lung disease and other abnormalities. The proband is a 4-year-old girl followed by clinical genetics since birth. Pregnancy history was unremarkable. She was born at 38 weeks gestation, following spontaneous rupture of membranes, withApgars of 8 and 9 at 1 and 5min respectively. She presented to the hospital at 2 months, due to a cough that developed into respiratory collapse. At that time, the patient was found to have apatent ductus arteriosus and coarctation of the aorta. Surgery was performed to correct the heart defects, but the lung disease persisted. Lung biopsy showed emphysematous changes, consistent with a congenital cystic lung disease. The patient has had recurrent respiratory infections since that time. Subsequent investigation for gastroesophageal reflux showed nonrotation of the bowel (duodenum is completely straight, small bowel is on the left side of the abdomen, large bowel is on the right). This finding is thought to be asymptomatic and probably unrelated to the gastroesophageal reflux. Recurrent urinary tract infections were investigated, and renal ultrasound showed dilated calyces in the left kidney, and dilation of the left ureter. The girl’s mother reported that her dentist noted that both of the proband’s lateral incisors have failed to develop. The mother had noted both pupils to be constantly dilated and unchanging since age 6 weeks. Ophthalmologic exam at age4years best corrected visual acuity of 20/40 in eacheye. She was wearing bifocals of 1.50 þ1.25 axis 90 in each eye with mild mixed astigmatism. Although clycloplegic drops (1% cycloplentalate) did not alter the size of the pupils, the refractive error was found to be less myopic, suggesting the presence of some accommodative ability in each eye. Her near point of accommodation was later measured at age 5 years as reduced to 6 inches in each eye. Without reading glasses she could read only the equivalent of 20/80 in each eye. This was improved to 20/20 with a reading addition of þ3.00 S in her bifocals which she found useful. The pupils were round and symmetrical, measuring 7 mm in the horizontal plane and showed no movement, either constriction to light or dilation in the dark, on clinical examination. Therewas absence of the iris collarette, the pupillary marginal pigmentary ruff, and the area of thickening characteristic of the pupillary sphincter. The iris stroma surrounding the pupillary opening was without radial or concentric iris folds and without stromal crypts usually seen, i.e., the iris appeared flat. On the iris surface were diffusely scattered small pigment clusters. There were remnant strands of the pupillary membrane in some areas of the pupil and strands of pigment on the lens surface. These iris and pupillary changes were symmetrical in both eyes. Retinal exam was normal, without any unusual retinal vessel tortuosity in either eye. Her ocular exam remained unchanged when she was last seen at age 5 years. Motor development was initially somewhat delayed, as the proband first walked at 17 months. Currently, her development is age appropriate, apart from delayed expressive language for which she is receiving speech therapy. Past investigations for the proband include an immunology work-up, sweat test for cystic fibrosis, alpha-1-antitrypsin analysis, a karyotype and FISH for microdeletion 22q11. The results of all were either normal or negative. Due to the spontaneous rupture of the membranes at 38 weeks, dilation of the left ureter, mild joint laxity and soft skin, a skin biopsy has been arranged to investigate the possibility of a collagen tissue disorder from skin fibrobalsts. No abnormality was detected in type I and type III procollagen and collagen. Physical examat 4years and7months of age showedanalert and active young girl. Her head circumference was below the 3rd centile at 47.5 cm. Her height was at the 10th centile at 98 cm, and weight was between the 10th and 25th centile at 15.4 kg. She was noted to have mild joint laxity and soft skin. She had nasal tone. Her philtrum was short, her nares anteverted, and she had an attached frenulum of the upper lip. No other dysmorphic features were noted. Review of the family history showed that the parents are nonconsanguineous and of English and Scottish ancestry. The proband has two older sisters who are normal. The proband’s father has a history of asthma and adult onset epilepsy. The maternal grandmother had spinocerebellar ataxia of an unknown type. There is no family history of developmental delay or heart defects. No other relatives had fixed large pupils although a paternal cousin had low vision and eyes that ‘‘jumped all over,’’ presumably nystagmus. Our proband exhibits both of the features described in the cases of Buys et al. [1993] and Graf and Jungherr [2002] *Correspondence to: Ahmad S. Teebi, MD, Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada. E-mail: ahmad.teebi@sickkids.ca

Mosaic tetrasomy 5p resulting from an isochromosome 5p marker chromosome: case report and review of literature.

We report on the fifth case, and oldest reported patient, of an individual affected with mosaic tetrasomy 5p resulting from an isochromosome 5p [i(5)(p10)] marker chromosome. A syndrome of mosaic tetrasomy 5p is defined, and includes the following features seen in the reported cases: developmental delay, seizures, ventriculomegaly (other brain anomalies), small stature/growth delay and mosaic pigmentary skin changes. Other findings include various dysmorphic facial features as well as hand and foot anomalies. This syndrome is likely more common than suggested in the literature, as the clinical presentation can be variable, and the chromosome anomaly is unlikely to be found on routine karyotype of peripheral blood lymphocytes. The i(5)(p10) marker chromosome is found only as a mosaic anomaly, with levels ranging from 0% to 10% in cultured lymphocytes to 12–85% in cultured skin fibroblasts. Microarray analysis performed on unstimulated lymphocytes from the patient in this report did not detect any evidence of the chromosome abnormality, indicating that this methodology may not be useful as a diagnostic tool in this disorder. Diagnosis of the mosaic tetrasomy 5p syndrome will rely on good clinical assessment, and appropriate cytogenetic studies, including analysis of skin fibroblasts. A child with unexplained developmental delay, seizures, hypotonia, and ventriculomegaly with or without dysmorphic features should be assessed carefully for pigmentary changes of the skin. If a diagnosis of mosaic 5p tetrasomy is suspected, karyotype of cultured fibroblasts in addition to routine cytogenetic analysis, to look for this marker chromosome is warranted.

An autosomal recessive form of Alagille-like syndrome that is not linked to JAG1

Purpose: Alagille syndrome is an autosomal dominant condition characterized by a paucity of interlobular bile ducts and chronic cholestasis, cardiac disease, skeletal abnormalities, ocular abnormalities, and characteristic facies. Most cases harbor a mutation in JAG1. We describe a large consanguineous family with five individuals affected with an Alagille-like syndrome that appears to be autosomal recessive. Our objective was to characterize the disorder clinically and determine whether affected individuals had inherited a mutation in JAG1.Methods: Clinical data were obtained through questioning and patient chart review. Linkage analysis using microsatellite markers was used to assess the possibility of a JAG1 mutation.Results: The clinical phenotype of patients was not entirely consistent with classic Alagille syndrome. All affected individuals had neonatal cholestasis with intrahepatic bile duct paucity, with three having pulmonary stenosis, but the presentation was unusually uniform and severe in childhood. There was no evidence of posterior embryotoxon or vertebral anomalies. Cardiac abnormalities were inconsistent between patients. Most significantly, the pedigree suggested an autosomal recessive form of inheritance. Linkage analysis excluded a mutation in JAG1.Conclusions: We have identified a kindred with an Alagille-like syndrome with an autosomal recessive form of inheritance not caused by a mutation in JAG1.

Novel splice-site mutation in ATP8B1 results in atypical Progressive Familial Intrahepatic Cholestasis Type 1

Our objective was to identify the molecular genetic basis of an Alagille‐like condition not linked to JAG1 or NOTCH2 in two related sibships.