Detection of elusive DNA copy-number variations in hereditary disease and cancer through the use of noncoding and off-target sequencing reads.

Copy-number variants (CNVs) play a substantial role in the molecular pathogenesis of hereditary disease and cancer, as well as in normal human interindividual variation. However, they are still rather difficult to identify in mainstream sequencing projects, especially involving exome sequencing, because they often occur in DNA regions that are not targeted for analysis. To overcome this problem, we developed OFF-PEAK, a user-friendly CNV detection tool that builds on a denoising approach and the use of "off-target" DNA reads, which are usually discarded by sequencing pipelines. We benchmarked OFF-PEAK on data from targeted sequencing of 96 cancer samples, as well as 130 exomes of individuals with inherited retinal disease from three different populations. For both sets of data, OFF-PEAK demonstrated excellent performance (>95% sensitivity and >80% specificity vs. experimental validation) in detecting CNVs from in silico data alone, indicating its immediate applicability to molecular diagnosis and genetic research.

Uniparental Disomy as a Mechanism for Combined Oxidative Phosphorylation Deficiency Associated with MRPS34 Gene.

INTRODUCTION: Mitochondrial oxidative phosphorylation (OXPHOS) is a cellular process that generates most of the cellular energy required by the body. Disorders affecting OXPHOS are multisystem diseases caused by pathogenic variants in more than 50 genes. In 2017, biallelic variants in the MRPS34 gene were shown to cause combined oxidative phosphorylation deficiency type 32 (COPD32) (OMIM#617664); however, only 7 patients have been reported in the literature up to this moment. COPD32 is characterized mainly by a severe Leigh-like syndrome. METHODS: Whole-exome sequencing identified a homozygous pathogenic variant in the MRPS34 gene, c.322-10G>A. Only the mother was heterozygous for this variant. SNP-array analysis was performed, which revealed a region of absence of heterozygosity in variant 16q with 9.8Mb, compatible with maternal uniparental disomy. RESULTS/CASE REPORT: We report the case of an 18-year-old female with unremarkable family history. The pregnancy was complicated by oligohydramnios, and the neonatal period was unremarkable. She evolved with low weight, mild-moderate developmental delay/intellectual disability, and hypogonadotropic hypogonadism. On examination, she had slender habitus, joint laxity, and kyphoscoliosis. The cardiac evaluation was normal, and the head MRI showed bilateral olivary nucleus degeneration that was not confirmed subsequently. Extensive metabolic studies documented only mild lactate and pyruvate elevation, and the chromosomal microarray was normal. CONCLUSION: We have reported the case of the first patient with COPD32 due to partial maternal uniparental disomy of chromosome 16, being first in Portugal and seventh in the literature. Contrarily to previous patients, who died in the first months of life or survived with severe DD/ID, and had a Leigh-like syndrome, this case is significantly milder, contributing to a better characterization of the phenotypic spectrum. Recurrence risk is unexpectedly low in this instance. This case illustrates the importance of segregation analysis in patients with homozygous recessive mutations.

The first genetic landscape of inherited retinal dystrophies in Portuguese patients identifies recurrent homozygous mutations as a frequent cause of pathogenesis.

Inherited retinal diseases (IRDs) are a group of ocular conditions characterized by an elevated genetic and clinical heterogeneity. They are transmitted almost invariantly as monogenic traits. However, with more than 280 disease genes identified so far, association of clinical phenotypes with genotypes can be very challenging, and molecular diagnosis is essential for genetic counseling and correct management of the disease. In addition, the prevalence and the assortment of IRD mutations are often population-specific. In this work, we examined 230 families from Portugal, with individuals suffering from a variety of IRD diagnostic classes (270 subjects in total). Overall, we identified 157 unique mutations (34 previously unreported) in 57 distinct genes, with a diagnostic rate of 76%. The IRD mutational landscape was, to some extent, different from those reported in other European populations, including Spanish cohorts. For instance, the EYS gene appeared to be the most frequently mutated, with a prevalence of 10% among all IRD cases. This was, in part, due to the presence of a recurrent and seemingly founder mutation involving the deletion of exons 13 and 14 of this gene. Moreover, our analysis highlighted that as many as 51% of our cases had mutations in a homozygous state. To our knowledge, this is the first study assessing a cross-sectional genotype-phenotype landscape of IRDs in Portugal. Our data reveal a rather unique distribution of mutations, possibly shaped by a small number of rare ancestral events that have now become prevalent alleles in patients.

Upper limb phocomelia: A prenatal case of thrombocytopenia-absent radius (TAR) syndrome illustrating the importance of chromosomal microarray in limb reduction defects.

OBJECTIVE: To describe the ultrasonographic, pathologic and molecular findings in a fetus with TAR syndrome, and to illustrate the contribution of chromosomal microarray analysis (CMA) to the etiological investigation of fetal upper limb reduction defects. CASE REPORT: A 35-year-old woman was referred for Genetic Counseling after pregnancy termination for severe upper limb bilateral phocomelia detected in the second trimester. Fetal autopsy showed severe shortening of the arms and forearms. The fetal skeletal survey confirmed the absence of the radii, ulnae and humeri. CMA revealed an interstitial deletion in 1q21 including the RBM8A gene. Subsequent Sanger sequencing of this gene identified a hypomorphic mutant allele, c.-21G > A, confirming the diagnosis of TAR syndrome. CONCLUSION: The differential diagnosis of upper limb defects is broad. Identification of their cause is essential for adequate genetic counseling including prognosis and recurrence risk estimation. CMA should be considered in fetuses with upper limb reduction defects, especially when the thumbs are present.

Genetics of personalized medicine: cancer and rare diseases.

The 21st annual meeting of the Portuguese Society of Human Genetics (SPGH), organized by Luísa Romão, Ana Sousa and Rosário Pinto Leite, was held in Caparica, Portugal, from the 16th to the 18th of November 2017. Having entered an era in which personalized medicine is emerging as a paradigm for disease diagnosis, treatment and prevention, the program of this meeting intended to include lectures by leading national and international scientists presenting exceptional findings on the genetics of personalized medicine. Various topics were discussed, including cancer genetics, transcriptome dynamics and novel therapeutics for cancers and rare disorders that are designed to specifically target molecular alterations in individual patients. Several panel discussions were held to emphasize (ethical) issues associated with personalized medicine, including genetic cancer counseling.

Overproduction, crystallization and preliminary X-ray characterization of Abn2, an endo-1,5-alpha-arabinanase from Bacillus subtilis.

Two Bacillus subtilis extracellular endo-1,5-alpha-L-arabinanases, AbnA and Abn2, belonging to glycoside hydrolase family 43 have been identified. The recently characterized Abn2 protein hydrolyzes arabinan and has low identity to other reported 1,5-alpha-L-arabinanases. Abn2 and its selenomethionine (SeMet) derivative have been purified and crystallized. Crystals appeared in two different space groups: P1, with unit-cell parameters a = 51.9, b = 57.6, c = 86.2 A, alpha = 82.3, beta = 87.9, gamma = 63.6 degrees , and P2(1)2(1)2(1), with unit-cell parameters a = 57.9, b = 163.3, c = 202.0 A. X-ray data have been collected for the native and the SeMet derivative to 1.9 and 2.7 A resolution, respectively. An initial model of Abn2 is being built in the SeMet-phased map.