Rowdy Meijer

Mutations in BICD2, which Encodes a Golgin and Important Motor Adaptor, Cause Congenital Autosomal-Dominant Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a heterogeneous group of neuromuscular disorders caused by degeneration of lower motor neurons. Although functional loss of SMN1 is associated with autosomal-recessive childhood SMA, the genetic cause for most families affected by dominantly inherited SMA is unknown. Here, we identified pathogenic variants in bicaudal D homolog 2 (Drosophila) (BICD2) in three families afflicted with autosomal-dominant SMA. Affected individuals displayed congenital slowly progressive muscle weakness mainly of the lower limbs and congenital contractures. In a large Dutch family, linkage analysis identified a 9q22.3 locus in which exome sequencing uncovered c.320C>T (p.Ser107Leu) in BICD2. Sequencing of 23 additional families affected by dominant SMA led to the identification of pathogenic variants in one family from Canada (c.2108C>T [p.Thr703Met]) and one from the Netherlands (c.563A>C [p.Asn188Thr]). BICD2 is a golgin and motor-adaptor protein involved in Golgi dynamics and vesicular and mRNA transport. Transient transfection of HeLa cells with all three mutant BICD2 cDNAs caused massive Golgi fragmentation. This observation was even more prominent in primary fibroblasts from an individual harboring c.2108C>T (p.Thr703Met) (affecting the C-terminal coiled-coil domain) and slightly less evident in individuals with c.563A>C (p.Asn188Thr) (affecting the N-terminal coiled-coil domain). Furthermore, BICD2 levels were reduced in affected individuals and trapped within the fragmented Golgi. Previous studies have shown that Drosophila mutant BicD causes reduced larvae locomotion by impaired clathrin-mediated synaptic endocytosis in neuromuscular junctions. These data emphasize the relevance of BICD2 in synaptic-vesicle recycling and support the conclusion that BICD2 mutations cause congenital slowly progressive dominant SMA.

Targeted next-generation sequencing of a 12.5 Mb homozygous region reveals ANO10 mutations in patients with autosomal-recessive cerebellar ataxia.

Autosomal-recessive cerebellar ataxias comprise a clinically and genetically heterogeneous group of neurodegenerative disorders. In contrast to their dominant counterparts, unraveling the molecular background of these ataxias has proven to be more complicated and the currently known mutations provide incomplete coverage for genotyping of patients. By combining SNP array-based linkage analysis and targeted resequencing of relevant sequences in the linkage interval with the use of next-generation sequencing technology, we identified a mutation in a gene and have shown its association with autosomal-recessive cerebellar ataxia. In a Dutch consanguineous family with three affected siblings a homozygous 12.5 Mb region on chromosome 3 was targeted by array-based sequence capture. Prioritization of all detected sequence variants led to four candidate genes, one of which contained a variant with a high base pair conservation score (phyloP score: 5.26). This variant was a leucine-to-arginine substitution in the DUF 590 domain of a 16K transmembrane protein, a putative calcium-activated chloride channel encoded by anoctamin 10 (ANO10). The analysis of ANO10 by Sanger sequencing revealed three additional mutations: a homozygous mutation (c.1150_1151del [p.Leu384fs]) in a Serbian family and a compound-heterozygous splice-site mutation (c.1476+1G>T) and a frameshift mutation (c.1604del [p.Leu535X]) in a French family. This illustrates the power of using initial homozygosity mapping with next-generation sequencing technology to identify genes involved in autosomal-recessive diseases. Moreover, identifying a putative calcium-dependent chloride channel involved in cerebellar ataxia adds another pathway to the list of pathophysiological mechanisms that may cause cerebellar ataxia.

Genotype-phenotype correlations in MYCN-related Feingold syndrome.

Feingold syndrome (FS) is the most frequent cause of familial syndromic gastrointestinal atresia and follows autosomal dominant inheritance. FS is caused by germline mutations in or deletions of the MYCN gene. Previously, 12 different heterozygous MYCN mutations and two deletions containing multiple genes including MYCN were described. All these mutations result in haploinsufficiency of both the canonical MYCN protein and the shorter isoform, ΔMYCN. We report 11 novel mutations including seven mutations in exon 2 that result in a premature termination codon (PTC) in the long MYCN transcript. Moreover, we have identified a PTC in exon 1 that only affects the ΔMYCN isoform, without a phenotypic effect. This suggests that mutations in only ΔMYCN do not contribute to the FS. Additionally, we found three novel deletions encompassing MYCN. Together with our previous report we now have a total of four missense mutations in the DNA binding domain, 19 PTCs of which six render the transcript subject to nonsense‐mediated decay (NMD), and five larger deletions in a total of 77 patients. We have reviewed the clinical features of these patients, and found that digital anomalies, e.g., brachymesophalangy and toe syndactyly, are the most consistent features, present in 100% and 97% of the patients, respectively. Small head circumference was present in 89% of the cases. Gastrointestinal atresia remains the most important major congenital anomaly (55%), but cardiac and renal anomalies are also frequent. We suggest that the presence of brachymesophalangy and toe syndactyly in combination with microcephaly is enough to justify MYCN analysis. Hum Mutat 29(9), 1125–1132, 2008.

Spectrum of p63 mutations in a selected patient cohort affected with ankyloblepharon-ectodermal defects-cleft lip/palate syndrome (AEC)†

Heterozygous mutations in the p63 gene underlie a group of at least seven allelic syndromes, including ankyloblepharon‐ectodermal defects‐cleft lip/palate syndrome (AEC) and Rapp Hodgkin syndrome (RHS), which involves varying degrees of ectodermal dysplasia, orofacial clefting and limb malformations. Mutations in the AEC and Rapp Hodgkin syndromes cluster in the 3′ end of the p63 gene. Previously reported mutations are mainly missense and frameshift mutations in exons 13 and 14, affecting the p63α‐specific SAM (sterile alpha motif) and TI (transactivation inhibitory) domains. A patient cohort affected by AEC syndrome was evaluated during International Research Symposium supported by the National Foundation for Ectodermal Dysplasias. Nineteen patients underwent full clinical evaluations and 18 had findings consistent with a diagnosis of AEC syndrome. These 19 patients, along with 5 additional relatives had genomic DNA analysis. Twenty‐one of the 24 participants from 12 families were found to have mutations in the p63 gene. Eleven different mutations were identified; 10 were novel mutations. Eight were missense mutations within the coding region of the SAM domain. Three other mutations were located in exon 14 sequences, which encode the TI domain. The effects of the mutations in the SAM and TI domains are poorly understood and functional studies are required to understand the pathological mechanisms. However, AEC and RHS mutations in the 5′ and 3′ ends of the p63 gene point towards a critical role of the ΔNp63α isoform for the AEC/RHS phenotype.

Mechanisms of natural gene therapy in dystrophic epidermolysis bullosa.

Revertant mosaicism has been reported in several inherited diseases, including the genetic skin fragility disorder epidermolysis bullosa (EB). Here, we describe the largest cohort of seven patients with revertant mosaicism and dystrophic EB (DEB), associated with mutations in the COL7A1 gene, and determine the underlying molecular mechanisms. We show that revertant mosaicism occurs both in autosomal dominantly and recessively inherited DEB. We found that null mutations resulting in complete loss of collagen VII and severe disease, as well as missense or splice-site mutations associated with some preserved collagen VII function and a milder phenotype, were corrected by revertant mosaicism. The mutation, subtype, and severity of the disease are thus not decisive for the presence of revertant mosaicism. Although collagen VII is synthesized and secreted by both keratinocytes and fibroblasts, evidence for reversion was only found in keratinocytes. The reversion mechanisms included back mutations/mitotic recombinations in 70% of the cases and second-site mutations affecting splicing in 30%. We conclude that revertant mosaicism is more common than previously assumed in patients with DEB, and our findings will have implications for future therapeutic strategies using the patients naturally corrected cells as a source for cell-based therapies.

Recurrent FXYD2 p.Gly41Arg mutation in patients with isolated dominant hypomagnesaemia

BACKGROUND Magnesium (Mg(2+)) is an essential ion for cell growth, neuroplasticity and muscle contraction. Blood Mg(2+) levels <0.7 mmol/L may cause a heterogeneous clinical phenotype, including muscle cramps and epilepsy and disturbances in K(+) and Ca(2+) homeostasis. Over the last decade, the genetic origin of several familial forms of hypomagnesaemia has been found. In 2000, mutations in FXYD2, encoding the γ-subunit of the Na(+)-K(+)-ATPase, were identified to cause isolated dominant hypomagnesaemia (IDH) in a large Dutch family suffering from hypomagnesaemia, hypocalciuria and chondrocalcinosis. However, no additional patients have been identified since then. METHODS Here, two families with hypomagnesaemia and hypocalciuria were screened for mutations in the FXYD2 gene. Moreover, the patients were clinically and genetically characterized. RESULTS We report a p.Gly41Arg FXYD2 mutation in two families with hypomagnesaemia and hypocalciuria. Interestingly, this is the same mutation as was described in the original study. As in the initial family, several patients suffered from muscle cramps, chondrocalcinosis and epilepsy. Haplotype analysis revealed an overlapping haplotype in all families, suggesting a founder effect. CONCLUSIONS The recurrent p.Gly41Arg FXYD2 mutation in two new families with IDH confirms that FXYD2 mutation causes hypomagnesaemia. Until now, no other FXYD2 mutations have been reported which could indicate that other FXYD2 mutations will not cause hypomagnesaemia or are embryonically lethal.

The inversa type of recessive dystrophic epidermolysis bullosa is caused by specific arginine and glycine substitutions in type VII collagen

Background The inversa type of recessive dystrophic epidermolysis bullosa (RDEB-I) is a rare variant of dystrophic epidermolysis bullosa, characterised by blistering in the body flexures, trunk, and mucosa. The cause of this specific distribution is unknown. So far, 20 COL7A1 genotypes have been described in RDEB-I and genotype–phenotype correlations have not been studied extensively. The aim of the study was to gain more insight into the pathophysiology of this intriguing RDEB-I phenotype. Methods Twenty Dutch and British RDEB-I patients, and full genotypes in 18 of them, were identified. The literature on RDEB-I genotypes was reviewed and an extensive genotype–phenotype correlation study for RDEB-I was conducted. Results All 20 patients had generalised blistering at birth and during early infancy. In most patients, the age of transition from generalised to inversa distribution was before the age of 4 years. A spectrum of disease severity, ranging from the mildest ‘mucosal only’ phenotype to the severest phenotype with limited acral involvement, was noted. The 29 genotypes of these RDEB-I patients and those reported in the literature revealed that RDEB-I is associated with specific recessive arginine and glycine substitutions in the triple helix domain of type VII collagen. Discussion and conclusion Why these specific arginine and glycine substitutions cause the inversa distribution remains unknown. It was not possible to identify clear differences in location and nature of substituting amino acids between these mutations and missense mutations causing other RDEB phenotypes. It is hypothesised that the higher skin temperature in the affected areas plays an important role in the pathophysiology of RDEB-I.

Expanding the clinical spectrum of MYCN-related feingold syndrome

Feingold syndrome (OMIM#164280) is an autosomal dominant disorder characterized by variable combinations of microcephaly, limb malformations, esophageal and duodenal atresias, and learning disability/mental retardation. Hand and foot abnormalities may include hypoplastic thumbs, clinodactyly of second and fifth fingers, syndactyly (characteristically between second and third and fourth and fifth toes), and shortened or absent middle phalanges [Feingold et al., 1997]. Cardiac and renal malformations, vertebral anomalies, and deafness have also been described in a minority of patients [Kellermayer et al., 2005]. As a result of the versatile clinical picture, this entity has also been reported as oculo-duodeno-esophageal-digital (ODED) syndrome, microcephaly-oculo-digitoesophageal-duodenal syndrome, and microcephalymesobrachyphalangy-tracheoesophageal-fistula (MMT) syndrome. However, some of the variable features of Feingold syndrome are included in ‘‘microcephalydigital abnormalities-normal intelligence’’ (MIM#602585), described as an independent, distinct condition [Kawame et al., 1997]. The critical region of Feingold syndrome was mapped to chromosome 2p23-p24 [Celli et al., 2003] and a recent article revealed MYCN (2p24.1), as a causative gene in this genetic entity [van Bokhoven et al., 2005]. These findings indicated that MYCN dosage is an important factor in early embryonic development andpostnatal brain growth. Indeed, studies in human fetal brain and mice suggest that MYCN is involved in embryonic limb, visceral organ, and nervous system development [Hirvonen et al., 1990; Moens et al., 1992; Stanton et al., 1992]. Here, we report on a family—expressing variable features of Feingold syndrome—who carry a novel mutation of MYCN. The boy with classical features of the syndrome and his mother and maternal grandmother, only possessing the clinical phenotype of ‘‘microcephaly-digital abnormalitiesnormal intelligence’’ disorder also carry the same pathogenic mutation. The 4-year-old boy was born as a first child from non-consanguineous parents. The pregnancy was complicated by maternal nephropathy. He was deliveredvaginally at 39weekswithApgars 4 and8 at 1 and 5 min, birth weight 3,300 g. Esophageal atresia (Vogt III/B type) was detected shortly after birth and was repaired surgically. Cranial ultrasound showed subependymal hemorrhage. He developed seizures and has been treated with anti-epileptics since the age of 10 months. At the age of 4 he could not walk independently and his developmental quotient was 59 (Budapest-Binet). On physical examination his length was 98 cm (10–25 pc), weight 13.5 kg (5 pc), and OFC 46 cm (< 2 SD). Microcephaly and scaphocephaly, epicanthal folds, down-slanting palpebral fissures, relatively large ears, bulbous nasal tip, and dental malocclusion could be observed (Fig. 1). He also had clinodactyly of the fifth finger of the hands, small distal phalanges of the thumbs, mild brachydactyly on the feet, contractures of the wrist, elbow, ankle, and knee, and increased muscle tone. Bullet-shaped distal phalanges of the first finger, middle phalanx hypoplasia of the fifth finger, and

Design and Validation of a Conformation Sensitive Capillary Electrophoresis-Based Mutation Scanning System and Automated Data Analysis of the More than 15 kbp-Spanning Coding Sequence of the SACS Gene

In this study, we developed and analytically validated a fully automated, robust confirmation sensitive capillary electrophoresis (CSCE) method to perform mutation scanning of the large SACS gene. This method facilitates a rapid and cost-effective molecular diagnosis of autosomal recessive spastic ataxia of Charlevoix-Saguenay. Critical issues addressed during the development of the CSCE system included the position of a DNA variant relative to the primers and the CG-content of the amplicons. The validation was performed in two phases; a retrospective analysis of 32 samples containing 41 different known DNA variants and a prospective analysis of 20 samples of patients clinically suspected of having autosomal recessive spastic ataxia of Charlevoix-Saguenay. These 20 samples appeared to contain 73 DNA variants. In total, in 32 out of the 45 amplicons, a DNA variant was present, which allowed verification of the detection capacity during the validation process. After optimization of the original design, the overall analytical sensitivity of CSCE for the SACS gene was 100%, and the analytical specificity of CSCE was 99.8%. In conclusion, CSCE is a robust technique with a high analytical sensitivity and specificity, and it can readily be used for mutation scanning of the large SACS gene. Furthermore this technique is less time-consuming and less expensive, as compared with standard automated sequencing.

Adermatoglyphia, previously unrecognized manifestation in ADULT syndrome†‡

Adermatoglyphia, Previously Unrecognized Manifestation in ADULT Syndrome Hiram Larangeira de Almeida Jr.,* Patr ıcia Caspary, Rodrigo Pereira Duquia, Rowdy Meijer, and Maurice van Steensel Department of Dermatology, Santa Casa de Porto Alegre, Porto Alegre, Brazil Department of Dermatology, Federal University of Pelotas, Pelotas, RS, Brazil Department of Dermatology, Catholic University of Pelotas, Pelotas, RS, Brazil Department of Human Genetics, University of Nijmegen, Nijmegen, the Netherlands Department of Dermatology, University Hospital Maastricht, Maastricht, the Netherlands