Evaluation of a whole-exome sequencing pipeline and benchmarking of causal germline variant prioritizers.

Most causal variants of Mendelian diseases are exonic. Whole-exome sequencing (WES) has become the diagnostic gold standard, but causative variant prioritization constitutes a bottleneck. Here we assessed an in-house sample-to-sequence pipeline and benchmarked free prioritization tools for germline causal variants from WES data. WES of 61 unselected patients with a known genetic disease cause was obtained. Variant prioritizations were performed by diverse tools and recorded to obtain a diagnostic yield when the causal variant was present in the first, fifth, and 10th top rankings. A fraction of causal variants was not captured by WES (8.2%) or did not pass the quality control criteria (13.1%). Most of the applications inspected were unavailable or had technical limitations, leaving nine tools for complete evaluation. Exomiser performed best in the top first rankings, while LIRICAL led in the top fifth rankings. Based on the more conservative top 10th rankings, Xrare had the highest diagnostic yield, followed by a three-way tie among Exomiser, LIRICAL, and PhenIX, then followed by AMELIE, TAPES, Phen-Gen,  AIVar, and VarNote-PAT. Xrare, Exomiser, LIRICAL, and PhenIX are the most efficient options for variant prioritization in real patient WES data.

Disruption of a miR-29 binding site leading to COL4A1 upregulation causes pontine autosomal dominant microangiopathy with leukoencephalopathy.

OBJECTIVE: Cerebral small vessel disease (cSVD) is a heterogeneous group of disorders. Screening of known cSVD genes identifies the causative mutation in <15% of familial cSVD cases. We sought to identify novel causes of cSVD. METHODS: We used linkage analysis and exome sequencing to identify the causal mutation in a French cSVD family. The identified candidate gene was then screened in 202 cSVD unrelated probands, including 1 proband from the first reported pontine autosomal dominant microangiopathy with leukoencephalopathy (PADMAL) family. Sanger sequencing was used to confirm variants in all mutated probands and analyze their segregation in probands' relatives. Mutation consequences were assessed with luciferase reporter assays and real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: A candidate heterozygous variant located in a predicted miR-29 microRNA binding site, within the 3' untranslated region of COL4A1, was identified in the large French cSVD family. Five additional unrelated probands, including the PADMAL proband, harbored heterozygous variants in this microRNA binding site. Variants cosegregated with the affected phenotype, and cumulative logarithm of odds score reached 6.03, establishing linkage to this locus. A highly significant difference was observed when comparing the number of variants within this binding site in cases and controls (p = 1.77 × 10E-12). RT-qPCR analyses of patients' primary fibroblasts and luciferase reporter assays strongly favor an upregulation of COL4A1 mediated by disruption of miR-29 binding to its target site. Magnetic resonance imaging features were characterized by the presence of multiple pontine infarcts in all symptomatic mutation carriers. INTERPRETATION: Mutations upregulating COL4A1 expression lead to PADMAL, a severe early onset ischemic cSVD, distinct from the various phenotypes associated with COL4A1 missense glycine mutations. Ann Neurol 2016;80:741-753.

Heterozygous HTRA1 mutations are associated with autosomal dominant cerebral small vessel disease.

Cerebral small vessel disease represents a heterogeneous group of disorders leading to stroke and cognitive impairment. While most small vessel diseases appear sporadic and related to age and hypertension, several early-onset monogenic forms have also been reported. However, only a minority of patients with familial small vessel disease carry mutations in one of known small vessel disease genes. We used whole exome sequencing to identify candidate genes in an autosomal dominant small vessel disease family in which known small vessel disease genes had been excluded, and subsequently screened all candidate genes in 201 unrelated probands with a familial small vessel disease of unknown aetiology, using high throughput multiplex polymerase chain reaction and next generation sequencing. A heterozygous HTRA1 variant (R166L), absent from 1000 Genomes and Exome Variant Server databases and predicted to be deleterious by in silico tools, was identified in all affected members of the index family. Ten probands of 201 additional unrelated and affected probands (4.97%) harboured a heterozygous HTRA1 mutation predicted to be damaging. There was a highly significant difference in the number of likely deleterious variants in cases compared to controls (P = 4.2 × 10(-6); odds ratio = 15.4; 95% confidence interval = 4.9-45.5), strongly suggesting causality. Seven of these variants were located within or close to the HTRA1 protease domain, three were in the N-terminal domain of unknown function and one in the C-terminal PDZ domain. In vitro activity analysis of HTRA1 mutants demonstrated a loss of function effect. Clinical features of this autosomal dominant small vessel disease differ from those of CARASIL and CADASIL by a later age of onset and the absence of the typical extraneurological features of CARASIL. They are similar to those of sporadic small vessel disease, except for their familial nature. Our data demonstrate that heterozygous HTRA1 mutations are an important cause of familial small vessel disease, and that screening of HTRA1 should be considered in all patients with a hereditary small vessel disease of unknown aetiology.

A Novel Heterozygous Deletion Variant in KLOTHO Gene Leading to Haploinsufficiency and Impairment of Fibroblast Growth Factor 23 Signaling Pathway.

Hyperphosphatemia is commonly present in end-stage renal disease. Klotho (KL) is implicated in phosphate homeostasis since it acts as obligate co-receptor for the fibroblast growth factor 23 (FGF23), a major phosphaturic hormone. We hypothesized that genetic variation in the KL gene might be associated with alterations in phosphate homeostasis resulting in hyperphosphatemia. We performed sequencing for determining KL gene variants in a group of resistant hyperphosphatemic dialysis patients. In a 67-year-old female, blood DNA sequencing revealed a heterozygous deletion of a T at position 1041 (c.1041delT) in exon 2. This variation caused a frameshift with substitution of isoleucine for phenylalanine and introduction of a premature termination codon (p.Ile348Phefs*28). cDNA sequencing showed absence of deletion-carrier transcripts in peripheral blood mononuclear cells suggesting degradation of these through a nonsense-mediated RNA decay pathway. Experiments in vitro showed that p.Ile348Phefs*28 variant impaired FGF23 signaling pathway, indicating a functional inactivation of the gene. In the patient, serum levels of KL were 2.9-fold lower than the mean level of a group of matched dialysis subjects, suggesting a compromise in the circulating protein concentration due to haploinsufficiency. These findings provide a new loss-of-function variant in the human KL gene, suggesting that genetic determinants might be associated to clinical resistant hyperphosphatemia.

Further refinement of COL4A1 and COL4A2 related cortical malformations.

Mutations in COL4A1 have been reported in schizencephaly and porencephaly combined with microbleeds or calcifications, often associated with ocular and renal abnormalities, myopathy, elevated creatine kinase levels and haemolytic anaemia. In this study, we aimed to clarify the phenotypic spectrum of COL4A1/A2 mutations in the context of cortical malformations that include schizencephaly, polymicrogyria and/or heterotopia. METHODS: We screened for COL4A1/A2 mutations in 9 patients with schizencephaly and/or polymicrogyria suspected to be caused by vascular disruption and leading to a cerebral haemorrhagic ischaemic event. These included 6 cases with asymmetrical or unilateral schizencephaly and/or polymicrogyria and 3 cases with bilateral schizencephaly. RESULTS: One de novo missense COL4A1 mutation (c.3715 G > A, p.(Gly1239Arg)) and two COL4A2 mutations were found, respectively in one familial case (c.4129G > A, p.(Gly1377Arg)) and one sporadic patient (c.1776+1G > A). In three other cases, COL4A1 variants of unknown significance were identified. None of our patients demonstrated neuromuscular or hematological anomalies. Brain malformations included a combination of schizencephaly, mainly asymmetrical, with porencephaly or ventriculomegaly (3/3 mutated patients). We did not observe microbleeds or microcalcifications in any of our cases, hence we do not believe that they represent a distinctive feature of COL4A1/A2 mutations. CONCLUSIONS: Our study further emphasizes the need to search for both COL4A1 and COL4A2 mutations in children presenting with uni- or bilateral polymicrogyria with schizencephaly, even in the absence of intracranial microbleeds, calcification or associated systemic features.

PDGFB partial deletion: a new, rare mechanism causing brain calcification with leukoencephalopathy.

Idiopathic basal ganglia calcification (IBGC) is a progressive cerebral disorder with diverse motor, cognitive, and psychiatric expression. It is inherited as an autosomal dominant trait. Three IBGC-causing genes have been identified in the past 2 years: SLC20A2, PDGFRB, and PDGFB. Biological and genetic evidence showed that loss of function of either SLC20A2 or the PDGFB/PDGFRB pathway was the mechanism underlying calcification in patients with a mutation. Recently, in a study focusing on SLC20A2, a large deletion at this locus was reported. No study has systematically searched for copy number variants (CNV) involving these three genes. We designed a quantitative PCR assay of multiple short fluorescent fragments (QMPSF) to detect CNVs involving one of these three genes in a single assay. Among the 27 unrelated patients from our IBGC case series with no mutation in SLC20A2, PDGFRB, and PDGFB, we identified in one patient a heterozygous partial deletion involving exons 2 to 5 of PDGFB. This patient exhibited both strio-pallido-dentate calcification and white matter hyperintensity of presumed vascular origin, associated with mood disorder, subtle cognitive decline, and gait disorder. We confirmed by RT-PCR experiments that the allele carrying the deletion was transcribed. The resulting cDNA lacks sequence for several critical functional domains of the protein. Intragenic deletion of PDGFB is a new and rare mechanism causing IBGC. CNVs involving the three IBGC-causing genes should be investigated in patients with no point mutation.