Andrew C. Orr

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.

Mutations in Centrosomal Protein CEP152 in Primary Microcephaly Families Linked to MCPH4

Primary microcephaly is a rare condition in which brain size is substantially diminished without other syndromic abnormalities. Seven autosomal loci have been genetically mapped, and the underlying causal genes have been identified for MCPH1, MCPH3, MCPH5, MCPH6, and MCPH7 but not for MCPH2 or MCPH4. The known genes play roles in mitosis and cell division. We ascertained three families from an Eastern Canadian subpopulation, each with one microcephalic child. Homozygosity analysis in two families using genome-wide dense SNP genotyping supported linkage to the published MCPH4 locus on chromosome 15q21.1. Sequencing of coding exons of candidate genes in the interval identified a nonconservative amino acid change in a highly conserved residue of the centrosomal protein CEP152. The affected children in these two families were both homozygous for this missense variant. The third affected child was compound heterozygous for the missense mutation plus a second, premature-termination mutation truncating a third of the protein and preventing its localization to centrosomes in transfected cells. CEP152 is the putative mammalian ortholog of Drosphila asterless, mutations in which affect mitosis in the fly. Published data from zebrafish are also consistent with a role of CEP152 in centrosome function. By RT-PCR, CEP152 is expressed in the embryonic mouse brain, similar to other MCPH genes. Like some other MCPH genes, CEP152 shows signatures of positive selection in the human lineage. CEP152 is a strong candidate for the causal gene underlying MCPH4 and may be an important gene in the evolution of human brain size.

Mutations in origin recognition complex gene ORC4 cause Meier-Gorlin syndrome

Meier-Gorlin syndrome is a rare autosomal recessive genetic condition whose primary clinical hallmarks include small stature, small external ears and small or absent patellae. Using marker-assisted mapping in multiple families from a founder population and traditional coding exon sequencing of positional candidate genes, we identified three different mutations in the gene encoding ORC4, a component of the eukaryotic origin recognition complex, in five individuals with Meier-Gorlin syndrome. In two such individuals that were negative for mutations in ORC4, we found potential mutations in ORC1 and CDT1, two other genes involved in origin recognition. ORC4 is well conserved in eukaryotes, and the yeast equivalent of the human ORC4 missense mutation was shown to be pathogenic in functional assays of cell growth. This is the first report, to our knowledge, of a germline mutation in any gene of the origin recognition complex in a vertebrate organism.

Mutations in the UBIAD1 Gene, Encoding a Potential Prenyltransferase, Are Causal for Schnyder Crystalline Corneal Dystrophy

Schnyder crystalline corneal dystrophy (SCCD, MIM 121800) is a rare autosomal dominant disease characterized by progressive opacification of the cornea resulting from the local accumulation of lipids, and associated in some cases with systemic dyslipidemia. Although previous studies of the genetics of SCCD have localized the defective gene to a 1.58 Mbp interval on chromosome 1p, exhaustive sequencing of positional candidate genes has thus far failed to reveal causal mutations. We have ascertained a large multigenerational family in Nova Scotia affected with SCCD in which we have confirmed linkage to the same general area of chromosome 1. Intensive fine mapping in our family revealed a 1.3 Mbp candidate interval overlapping that previously reported. Sequencing of genes in our interval led to the identification of five putative causal mutations in gene UBIAD1, in our family as well as in four other small families of various geographic origins. UBIAD1 encodes a potential prenyltransferase, and is reported to interact physically with apolipoprotein E. UBIAD1 may play a direct role in intracellular cholesterol biochemistry, or may prenylate other proteins regulating cholesterol transport and storage.

Human monogenic disorders — a source of novel drug targets

The decrease in new drug applications and approvals over the past several years results from an underlying crisis in drug target identification and validation. Model organisms are being used to address this problem, in combination with novel approaches such as the International HapMap Project. What has been underappreciated is that discovery of new drug targets can also be revived by traditional Mendelian genetics. A large fraction of the human gene repertoire remains phenotypically uncharacterized, and is likely to encode many unanticipated and novel phenotypes that will be of interest to pharmaceutical and biotechnological drug developers.

Mutation in Pyrroline-5-Carboxylate Reductase 1 Gene in Families with Cutis Laxa Type 2

Autosomal-recessive cutis laxa type 2 (ARCL2) is a multisystem disorder characterized by the appearance of premature aging, wrinkled and lax skin, joint laxity, and a general developmental delay. Cutis laxa includes a family of clinically overlapping conditions with confusing nomenclature, generally requiring molecular analyses for definitive diagnosis. Six genes are currently known to mutate to yield one of these related conditions. We ascertained a cohort of typical ARCL2 patients from a subpopulation isolate within eastern Canada. Homozygosity mapping with high-density SNP genotyping excluded all six known genes, and instead identified a single homozygous region near the telomere of chromosome 17, shared identically by state by all genotyped affected individuals from the families. A putative pathogenic variant was identified by direct DNA sequencing of genes within the region. The single nucleotide change leads to a missense mutation adjacent to a splice junction in the gene encoding pyrroline-5-carboxylate reductase 1 (PYCR1). Bioinformatic analysis predicted a pathogenic effect of the variant on splice donor site function. Skipping of the associated exon was confirmed in RNA from blood lymphocytes of affected homozygotes and heterozygous mutation carriers. Exon skipping leads to deletion of the reductase functional domain-coding region and an obligatory downstream frameshift. PYCR1 plays a critical role in proline biosynthesis. Pathogenicity of the genetic variant in PYCR1 is likely, given that a similar clinical phenotype has been documented for mutation carriers of another proline biosynthetic enzyme, pyrroline-5-carboxylate synthase. Our results support a significant role for proline in normal development.

Exfoliation syndrome: clinical and genetic features.

We have ascertained a large number of individuals and families with exfoliation syndrome in order to clarify the disorder’s mode of inheritance. Patients with exfoliation syndrome and their relatives were recruited from the practices of a group of ophthalmologists in Maritime Canada. The degree to which the subjects were affected was graded according to a standardized 1–4-point clinical scheme. Pedigrees were constructed from information supplied by family members and from genealogical sources. A total of 782 patients and relatives participated, of whom 467 were definitely affected. The mean age of affected males and females did not differ significantly, but females appeared to be more severely affected at ascertainment than males. More than half of the affected subjects had definite exfoliation in only one eye. Approximately 30 multiplex families were discovered, including one containing 23 affected members among a total of 137 examined individuals that constitutes the largest exfoliative pedigree thus far described. We observed well-documented paternal transmission of the trait, a finding that has not to our knowledge been previously reported. Clustering of cases in the families provides evidence for the involvement of genetic factors. The possibility of homozygosity is suggested in a few patients by the earlier or more frequent presentation of the disorder in the offspring of two affected parents or consanguineous pairings. Although a multifactorial mode of inheritance cannot be excluded, exfoliation syndrome appears to be inherited as an autosomal dominant trait whose late onset and incomplete penetrance poses a significant but not insuperable obstacle to pedigree construction.

Mutation in the gene encoding ubiquitin ligase LRSAM1 in patients with Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease (CMT) represents a family of related sensorimotor neuropathies. We studied a large family from a rural eastern Canadian community, with multiple individuals suffering from a condition clinically most similar to autosomal recessive axonal CMT, or AR-CMT2. Homozygosity mapping with high-density SNP genotyping of six affected individuals from the family excluded 23 known genes for various subtypes of CMT and instead identified a single homozygous region on chromosome 9, at 122,423,730–129,841,977 Mbp, shared identical by state in all six affected individuals. A homozygous pathogenic variant was identified in the gene encoding leucine rich repeat and sterile alpha motif 1 (LRSAM1) by direct DNA sequencing of genes within the region in affected DNA samples. The single nucleotide change mutates an intronic consensus acceptor splicing site from AG to AA. Direct analysis of RNA from patient blood demonstrated aberrant splicing of the affected exon, causing an obligatory frameshift and premature truncation of the protein. Western blotting of immortalized cells from a homozygous patient showed complete absence of detectable protein, consistent with the splice site defect. LRSAM1 plays a role in membrane vesicle fusion during viral maturation and for proper adhesion of neuronal cells in culture. Other ubiquitin ligases play documented roles in neurodegenerative diseases. LRSAM1 is a strong candidate for the causal gene for the genetic disorder in our kindred.

Attitudes of parents toward the return of targeted and incidental genomic research findings in children.

Purpose:We describe parental attitudes toward the return of targeted and incidental genomic research results in the setting of high-risk pediatric cancer and inherited childhood diseases.Methods:A validated 36-item questionnaire was mailed to participants in three large-scale genome research consortia examining attitudes toward receipt of genomic research results and the influence of certainty, severity, and onset of the condition, in addition to responsibilities to extended family and provision of results even after death of the proband.Results:Of the 563 participants who were sent questionnaires, 362 (64%) responded. Most of them stated a positive right to receive results related to the target condition (97%) or to incidental findings (86%); no difference was found in results between participants with cancer and those with orphan diseases. Furthermore, 92% indicated that genomic research for childhood-onset conditions should occur. The majority wanted incidental results predicting susceptibility even to untreatable fatal conditions (83%), to multiple conditions (87%), or to those with uncertain impact (70%). Most felt sibling genomic results showing serious conditions, whether treatable (93%) or not (88%), and/or results discovered after death of the proband should be shared with family (74%).Conclusion:Many parents of children in pediatric genomic research indicated a strong desire to receive a broader range of results than is described in consensus recommendations. Clear delineation of what will be offered should be established at the time of consent.Genet Med 16 8, 633–640.Genetics in Medicine (2014); 16 8, 633–640. doi:10.1038/gim.2013.201

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.