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In 2016, guidelines for diagnostic Next Generation Sequencing (NGS) have been published by EuroGentest in order to assist laboratories in the implementation and accreditation of NGS in a diagnostic setting. These guidelines mainly focused on Whole Exome Sequencing (WES) and targeted (gene panels) sequencing detecting small germline variants (Single Nucleotide Variants (SNVs) and insertions/deletions (indels)). Since then, Whole Genome Sequencing (WGS) has been increasingly introduced in the diagnosis of rare diseases as WGS allows the simultaneous detection of SNVs, Structural Variants (SVs) and other types of variants such as repeat expansions. The use of WGS in diagnostics warrants the re-evaluation and update of previously published guidelines. This work was jointly initiated by EuroGentest and the Horizon2020 project Solve-RD. Statements from the 2016 guidelines have been reviewed in the context of WGS and updated where necessary. The aim of these recommendations is primarily to list the points to consider for clinical (laboratory) geneticists, bioinformaticians, and (non-)geneticists, to provide technical advice, aid clinical decision-making and the reporting of the results.

Using a genome-wide single nucleotide polymorphism (SNP) panel, we observed population structure in a diverse group of Europeans and European Americans. Under a variety of conditions and tests, there is a consistent and reproducible distinction between \"northern\" and \"southern\" European population groups: most individual participants with southern European ancestry (Italian, Spanish, Portuguese, and Greek) have >85% membership in the \"southern\" population; and most northern, western, eastern, and central Europeans have >90% in the \"northern\" population group. Ashkenazi Jewish as well as Sephardic Jewish origin also showed >85% membership in the \"southern\" population, consistent with a later Mediterranean origin of these ethnic groups. Based on this work, we have developed a core set of informative SNP markers that can control for this partition in European population structure in a variety of clinical and genetic studies.

The past decade has seen remarkable advances in defining the molecular mechanisms underlying formation of the embryonic left right (LR) axis. This information is slowly transforming our understanding of human birth defects that are caused by disturbed LR axis patterning. Reversals, isomerisms, or segmental discordances of thoraco-abdominal organ position, that is, classic heterotaxy, clearly indicate embryonic disruption of normal LR patterning. Other isolated birth defects, particularly cardiovascular malformations, may be caused by deficiencies in the same pathways. Here, we review the distinctive clinical features of human heterotaxias and try to summarize the known connections between them and the corresponding developmental pathways.

We and others have identified several hundred ancestry informative markers (AIMs) with large allele frequency differences between different major ancestral groups. For this study, a panel of 199 widely distributed AIMs was used to examine a diverse set of 796 DNA samples including self-identified European Americans, West Africans, East Asians, Amerindians, African Americans, Mexicans, Mexican Americans, Puerto Ricans and South Asians. Analysis using a Bayesian clustering algorithm (STRUCTURE) showed grouping of individuals with similar ethnic identity without any identifier other than the AIMs genotyping and showed admixture proportions that clearly distinguished different individuals of mixed ancestry. Additional analyses showed that, for the majority of samples, the predicted ethnic identity corresponded with the self-identified ethnicity at high probability (P > 0.99). Overall, the study demonstrates that AIMs can provide a useful adjunct to forensic medicine, pharmacogenomics and disease studies in which major ancestry or ethnic affiliation might be linked to specific outcomes.

Within the group of children with CHARGE association, there is clearly a subgroup with distinctive clinical characteristics that appears to have a recognisable syndrome. 6 Previously, a systematic scan for loss of heterozygosity using microsatellite markers in 10 such patients failed to identify a discernible submicroscopic deletion. 7 Although several candidate genes such as PITX2 8 and PAX2 9 have been investigated, no mutations have been identified in patients with CHARGE syndrome. METHODS The study sample includes 72 patients, of whom 43 have either four major criteria for CHARGE (coloboma, choanal atresia, characteristic ear abnormality, and cranial nerve dysfunction) or three major and three minor criteria (genital hypoplasia, developmental delay, cardiovascular malformations, growth deficiency, orofacial cleft, tracheo-oesopageal fistula, and characteristic face) as described by Blake et al. 10 This set of patients was used for all FISH analyses, DHPLC, and candidate gene sequencing.

Markers with large differences in allele frequencies between ethnicities provide ancestry information that can be applied to genetic studies. We identified over 100 biallelic ancestry informative markers (AIMs) with large allele frequency differences between European Americans (EA) and Pima Amerindians from laboratory and database screens. For 35 of these markers, Mayan, Yavapai and Quechuan Amerindians were genotyped and compared with EA and Pima allele frequencies. Markers with large allele frequency differences between EA and one Amerindian tribe showed only small differences between the Amerindian tribes. Examination of structure in individuals demonstrated a clear separation of subjects of European from those of Amerindian ancestry, and similarity between individuals from disparate Amerindian populations. The AIMs demonstrated the variation in ancestral composition of individual Mexican Americans, providing evidence of applicability in admixture mapping and in controlling for structure in association tests. In addition, a high percentage of single-nucleotide polymorphisms (SNPs) selected on the basis of large frequency differences between EA and Asian populations had large allele frequency differences between EA and Amerindians, suggesting an efficient method for greatly expanding AIMs for use in admixture mapping/structure analysis in Mexican Americans. Together, these data provide additional support for the practical application of admixture mapping in the Mexican American population.

We have generated mice with a null mutation at the Ada locus, which encodes the purine catabolic enzyme adenosine deaminase (ADA, EC 3.5.4.4). ADA-deficient fetuses exhibited hepatocellular impairment and died perinatally. Their lymphoid tissues were not largely affected. Accumulation of ADA substrates was detectable in ADA-deficient conceptuses as early as 12.5 days postcoitum, dramatically increasing during late in utero development, and is the likely cause of liver damage and fetal death. The results presented here demonstrate that ADA is important for the homeostatic maintenance of purines in mice.

Deficiency of iduronate-2-sulphatase (IDS) results in the X linked recessive lysosomal storage disorder Hunter syndrome. Determination of carrier status in families affected by this disorder has been performed using a variety of enzymatic tests. None of these tests has proved to be 100% effective at identifying carriers. The aim of this study was to perform carrier testing in a family affected by the disorder, where testing was complicated by the fact that no surviving affected subjects were available for study. Direct dye primer sequencing of PCR products was used to identify mixed bases in an obligate carrier. Two mixed bases were observed within exon VIII. The first base change (T-->A) at nucleotide position 1150 results in a missense mutation (H342Q), while the second base change (G-->T) at nucleotide position 1151 results in a nonsense mutation (G343X). Four additional female family members were screened for the same mutation. Using this approach it is possible to provide unambiguous information about a subject's carrier status and, unlike biochemical testing, this approach will be equally effective when applied to families with the mild form of this disorder.

Congenital Heart Disease (CHD) affects approximately 7-9 children per 1000 live births. Numerous genetic studies have established a role for rare genomic variants at the copy number variation (CNV) and single nucleotide variant level. In particular, the role of de novo mutations (DNM) has been highlighted in syndromic and non-syndromic CHD. To identify novel haploinsufficient CHD disease genes we performed an integrative analysis of CNVs and DNMs identified in probands with CHD including cases with sporadic thoracic aortic aneurysm (TAA). We assembled CNV data from 7,958 cases and 14,082 controls and performed a gene-wise analysis of the burden of rare genomic deletions in cases versus controls. In addition, we performed mutation rate testing for DNMs identified in 2,489 parent-offspring trios. Our combined analysis revealed 21 genes which were significantly affected by rare genomic deletions and/or constrained non-synonymous de novo mutations in probands. Fourteen of these genes have previously been associated with CHD while the remaining genes (FEZ1, MYO16, ARID1B, NALCN, WAC, KDM5B and WHSC1) have only been associated in singletons and small cases series, or show new associations with CHD. In addition, a systems level analysis revealed shared contribution of CNV deletions and DNMs in CHD probands, affecting protein-protein interaction networks involved in Notch signaling pathway, heart morphogenesis, DNA repair and cilia/centrosome function. Taken together, this approach highlights the importance of re-analyzing existing datasets to strengthen disease association and identify novel disease genes.

ABSTRACTBACKGROUND: Videokeratography may provide information for surgical correction of astigmatism after penetrating keratoplasty. We used a combination of wedge resection and relaxing incisions to treat high refractive astigmatism after penetrating keratoplasty.METHODS: Videokeratography using the normalized scale of the Topographic Modeling System was used as a guide in determining the location and the length of incisions and resections. Nine eyes were treated with both relaxing incisions and a wedge resection. All patients had more than 3.00 diopters (D) of refractive astigmatism. All patients were intolerant of spectacles or contact lenses. The depth of the corneal relaxing incisions was constant at 0.5 mm and the width of the corneal wedge resections was constant at 0.75 mm.RESULTS: The relaxing incisions produced flattening of the steeper meridian and the wedge resection produced steepening of the flatter meridian. The average preoperative keratometric astigmatism was 7.44 D (range, 3.50 to 11.00 D) and the average refractive astigmatism was 5.56 D (range, 4.00 to 8.00 D). The average preoperative spherical equivalent was 0.08 D (range, -7.00 to 4.25 D). Postoperatively, the average keratometric astigmatism was 2.97 D (range, 1.00 to 5.00 D) and the average refractive astigmatism was 2.58 D (range, 0.00 to 5.00 D). The average postoperative spherical equivalent refraction was -0.32 D.CONCLUSIONS: Combined corneal wedge resection and relaxing incisions appears to be effective in reducing high refractive astigmatism following corneal transplantation. [J Refract Surg. 1995;11:472-476.]