Michael F. Buckley

Next-generation genetic testing for retinitis pigmentosa

Molecular diagnostics for patients with retinitis pigmentosa (RP) has been hampered by extreme genetic and clinical heterogeneity, with 52 causative genes known to date. Here, we developed a comprehensive next‐generation sequencing (NGS) approach for the clinical molecular diagnostics of RP. All known inherited retinal disease genes (n = 111) were captured and simultaneously analyzed using NGS in 100 RP patients without a molecular diagnosis. A systematic data analysis pipeline was developed and validated to prioritize and predict the pathogenicity of all genetic variants identified in each patient, which enabled us to reduce the number of potential pathogenic variants from approximately 1,200 to zero to nine per patient. Subsequent segregation analysis and in silico predictions of pathogenicity resulted in a molecular diagnosis in 36 RP patients, comprising 27 recessive, six dominant, and three X‐linked cases. Intriguingly, De novo mutations were present in at least three out of 28 isolated cases with causative mutations. This study demonstrates the enormous potential and clinical utility of NGS in molecular diagnosis of genetically heterogeneous diseases such as RP. De novo dominant mutations appear to play a significant role in patients with isolated RP, having major implications for genetic counselling. Hum Mutat 33:963–972, 2012.

Dystrophin gene mutation location and the risk of cognitive impairment in Duchenne muscular dystrophy.

Background A significant component of the variation in cognitive disability that is observed in Duchenne muscular dystrophy (DMD) is known to be under genetic regulation. In this study we report correlations between standardised measures of intelligence and mutational class, mutation size, mutation location and the involvement of dystrophin isoforms. Methods and Results Sixty two male subjects were recruited as part of a study of the cognitive spectrum in boys with DMD conducted at the Sydney Childrens Hospital (SCH). All 62 children received neuropsychological testing from a single clinical psychologist and had a defined dystrophin gene (DMD) mutation; including DMD gene deletions, duplications and DNA point mutations. Full Scale Intelligence Quotients (FSIQ) in unrelated subjects with the same mutation were found to be highly correlated (r = 0.83, p = 0.0008), in contrast to results in previous publications. In 58 cases (94%) it was possible to definitively assign a mutation as affecting one or more dystrophin isoforms. A strong association between the risk of cognitive disability and the involvement of groups of DMD isoforms was found. In particular, improvements in the correlation of FSIQ with mutation location were identified when a new classification system for mutations affecting the Dp140 isoform was implemented. Significance These data represent one of the largest studies of FSIQ and mutational data in DMD patients and is among the first to report on a DMD cohort which has had both comprehensive mutational analysis and FSIQ testing through a single referral centre. The correlation between FSIQ results with the location of the dystrophin gene mutation suggests that the risk of cognitive deficit is a result of the cumulative loss of central nervous system (CNS) expressed dystrophin isoforms, and that correct classification of isoform involvement results in improved estimates of risk.

SLC29A3 gene is mutated in pigmented hypertrichosis with insulin-dependent diabetes mellitus syndrome and interacts with the insulin signaling pathway

Pigmented hypertrichotic dermatosis with insulin-dependent diabetes (PHID) syndrome is a recently described autosomal recessive disorder associated with predominantly antibody negative, insulin-dependent diabetes mellitus. In order to identify the genetic basis of PHID and study its relationship with glucose metabolism, we performed homozygosity mapping in five unrelated families followed by candidate gene sequencing. Five loss-of-function mutations were identified in the SLC29A3 gene which encodes a member of a highly conserved protein family that transports nucleosides, nucleobases and nucleoside analogue drugs, hENT3. We show that PHID is allelic with a related syndrome without diabetes mellitus, H syndrome. The interaction of SLC29A3 with insulin signaling pathways was then studied using an established model in Drosophila melanogaster. Ubiquitous knockdown of the Drosophila ortholog of hENT3, dENT1 is lethal under stringent conditions; whereas milder knockdown induced scutellar bristle phenotypes similar to those previously reported in the knockdown of the Drosophila ortholog of the Islet gene. A cellular growth assay showed a reduction of cell size/number which could be rescued or enhanced by manipulation of the Drosophila insulin receptor and its downstream signaling effectors, dPI3K and dAkt. In summary, inactivating mutations in SLC29A3 cause a syndromic form of insulin-dependent diabetes in humans and in Drosophila profoundly affect cell size/number through interactions with the insulin signaling pathway. These data suggest that further investigation of the role of SLC29A3 in glucose metabolism is a priority for diabetes research.

A genome-wide association study identifies susceptibility loci for nonsyndromic sagittal craniosynostosis near BMP2 and within BBS9

Sagittal craniosynostosis is the most common form of craniosynostosis, affecting approximately one in 5,000 newborns. We conducted, to our knowledge, the first genome-wide association study for nonsyndromic sagittal craniosynostosis (sNSC) using 130 non-Hispanic case-parent trios of European ancestry (NHW). We found robust associations in a 120-kb region downstream of BMP2 flanked by rs1884302 (P = 1.13 × 10−14, odds ratio (OR) = 4.58) and rs6140226 (P = 3.40 × 10−11, OR = 0.24) and within a 167-kb region of BBS9 between rs10262453 (P = 1.61 × 10−10, OR = 0.19) and rs17724206 (P = 1.50 × 10−8, OR = 0.22). We replicated the associations to both loci (rs1884302, P = 4.39 × 10−31 and rs10262453, P = 3.50 × 10−14) in an independent NHW population of 172 unrelated probands with sNSC and 548 controls. Both BMP2 and BBS9 are genes with roles in skeletal development that warrant functional studies to further understand the etiology of sNSC.

Mutations in the gene encoding the PML nuclear body protein Sp110 are associated with immunodeficiency and hepatic veno-occlusive disease

We describe mutations in the PML nuclear body protein Sp110 in the syndrome veno-occlusive disease with immunodeficiency, an autosomal recessive disorder of severe hypogammaglobulinemia, combined T and B cell immunodeficiency, absent lymph node germinal centers, absent tissue plasma cells and hepatic veno-occlusive disease. This is the first report of the involvement of a nuclear body protein in a human primary immunodeficiency and of high-penetrance genetic mutations in hepatic veno-occlusive disease.

Heterozygous mutations of FREM1 are associated with an increased risk of isolated metopic craniosynostosis in humans and mice

The premature fusion of the paired frontal bones results in metopic craniosynostosis (MC) and gives rise to the clinical phenotype of trigonocephaly. Deletions of chromosome 9p22.3 are well described as a cause of MC with variably penetrant midface hypoplasia. In order to identify the gene responsible for the trigonocephaly component of the 9p22.3 syndrome, a cohort of 109 patients were assessed by high-resolution arrays and MLPA for copy number variations (CNVs) involving 9p22. Five CNVs involving FREM1, all of which were de novo variants, were identified by array-based analyses. The remaining 104 patients with MC were then subjected to targeted FREM1 gene re-sequencing, which identified 3 further mutant alleles, one of which was de novo. Consistent with a pathogenic role, mouse Frem1 mRNA and protein expression was demonstrated in the metopic suture as well as in the pericranium and dura mater. Micro-computed tomography based analyses of the mouse posterior frontal (PF) suture, the human metopic suture equivalent, revealed advanced fusion in all mice homozygous for either of two different Frem1 mutant alleles, while heterozygotes exhibited variably penetrant PF suture anomalies. Gene dosage-related penetrance of midfacial hypoplasia was also evident in the Frem1 mutants. These data suggest that CNVs and mutations involving FREM1 can be identified in a significant percentage of people with MC with or without midface hypoplasia. Furthermore, we present Frem1 mutant mice as the first bona fide mouse model of human metopic craniosynostosis and a new model for midfacial hypoplasia.

Measurement of the clinical utility of a combined mutation detection protocol in carriers of Duchenne and Becker muscular dystrophy

Background: Recent methodological advances have improved the detection rate for dystrophin mutations, but there are no published studies that have measured the clinical utility of these protocols for carrier detection compared with conventional carrier testing protocols that use pedigree, serum creatine kinase levels and linkage analysis. Methods and subjects: The clinical utility of a combined mutation detection protocol was measured. It involved quantitative PCR procedures followed by DNA sequence analysis for the identification of dystrophin mutation carriers in 2101 women at risk of being carriers from 348 mutation-known Duchenne or Becker muscular dystrophy pedigrees. Results: The combined mutation detection protocol identified a mutation in 96% and 82% of index cases of Duchenne muscular dystrophy and Becker muscular dystrophy, respectively. An additional 692 (33%) potential carriers were correctly classified by the combined mutation detection protocol compared with pedigree, serum creatine kinase levels and linkage analysis. Significantly lower mutation carrier rates were identified in the mothers of isolated cases with deletion mutations than predicted from theoretical considerations, but these findings were not confirmed for duplication and DNA sequence mutations. Conclusions: There are significant clinical benefits to be gained from a combined mutation detection protocol for carrier detection. It is recommended that mutation-specific carrier frequencies for the different classes of dystrophin mutations should be taken into account in genetic counselling practice.

The frequency of founder mutations in the BRCA1, BRCA2, and APC genes in australian Ashkenazi Jews

Several studies have shown that Ashkenazi Jews in the United States and Israel have a high prevalence of the founder mutations BRCA1 185delAG, BRCA1 5382insC, BRCA2 6174delT, and APC I1307K at frequencies of 1.0–1.1%, 0.2–0.3%, 0.6–1.4%, and 6.1–7.0%, respectively. The objective of this study was to compare the prevalence of these alleles in the Australian Jewish population with that of U.S. Jews. Australian Jews have a different history of migration, with less opportunity for changes in allele frequency due to conversion or intermarriage with non‐Jewish Australians. The results obtained therefore can be used to assess whether U.S. data can be generalized to other Jewish populations.

Expanding the clinical spectrum of SLC29A3 gene defects

H syndrome and pigmented hypertrichosis with insulin dependent diabetes (PHID) are allelic autosomal recessive syndromes reported in the last year to be caused by mutations in the SLC29A3 gene, which encodes the equilibrative nucleoside transporter hENT3. Herein, we report three new patients from a single family who present with phenotypes that associate features of both PHID and H syndrome. Genetic analysis of the SLC29A3 gene revealed that two affected sisters are compound heterozygotes for the previously reported mutations p.G427S and p.G437R, while their nephew was homozygous for the p.G437R mutation. In addition to this intra-familial genetic heterogeneity, these patients demonstrate considerable phenotypic variability. One sister had clinical features consistent with classical PHID phenotype, while her nephews features were in keeping with the diagnosis of H syndrome. The second sister displayed the most severe phenotype which combined diagnostic features from both syndromes. This patient also had features not described previously, including severe seronegative polyarthritis involving large and small joints, and hypogonadotropic hypogonadism. These manifestations may be additional characteristics of the growing clinical spectrum of SLC29A3 defects. This report emphasizes the complex genotype phenotype correlation in SLC29A3 disorders and suggests that other factors are relevant to disease manifestations and severity.

Quantitative Trait Loci Modifying Cardiac Atrial Septal Morphology and Risk of Patent Foramen Ovale in the Mouse

Atrial septal defect (ASD) is a common congenital heart disease (CHD) occurring in 5 to 7 per 10 000 live births. Mutations in 5 human genes (NKX2.5, TBX5, GATA4, MYHC, ACTC) are known to cause dominant ASD, but these account for a minority of cases. Human and mouse data suggest that ASD exists in an anatomical continuum with milder septal variants patent foramen ovale (PFO) and atrial septal aneurysm, strongly associated with ischemic stroke and migraine. We have previously shown in inbred mice that the incidence of PFO strongly correlates with length of the interatrial septum primum, defining a quantitative trait underlying PFO risk. To better understand genetic causation of atrial septal abnormalities, we mapped quantitative trait loci (QTL) influencing septal morphology using mouse strains (QSi5 and 129T2/SvEms) maximally informative for PFO incidence and 3 quantitative septal anatomical traits including septum primum length. [QSi5×129T2/SvEms]F2 intercross animals (n=1437) were phenotyped and a whole genome scan performed at an average 17-cM interval. Statistical methodology scoring PFO as a binary phenotype was developed as a confirmatory mapping technique. We mapped 7 significant and 6 suggestive QTL modifying quantitative phenotypes, with 4 supported by binary analysis. Quantitative traits, although strongly associated with PFO (P<0.001), correlated poorly with each other and in all but 1 case QTL for different traits were nonoverlapping. Thus, multiple anatomical processes under separate genetic control contribute to risk of PFO. Our findings demonstrate the feasibility of modeling the genetic basis of common CHD using animal genetic and genomic technologies.