Copy Number Variant Analysis and Genome-wide Association Study Identify Loci with Large Effect for Vesicoureteral Reflux.

BACKGROUND: Vesicoureteral reflux (VUR) is a common, familial genitourinary disorder, and a major cause of pediatric urinary tract infection (UTI) and kidney failure. The genetic basis of VUR is not well understood. METHODS: A diagnostic analysis sought rare, pathogenic copy number variant (CNV) disorders among 1737 patients with VUR. A GWAS was performed in 1395 patients and 5366 controls, of European ancestry. RESULTS: Altogether, 3% of VUR patients harbored an undiagnosed rare CNV disorder, such as the 1q21.1, 16p11.2, 22q11.21, and triple X syndromes ((OR, 3.12; 95% CI, 2.10 to 4.54; P=6.35×10-8) The GWAS identified three study-wide significant and five suggestive loci with large effects (ORs, 1.41-6.9), containing canonical developmental genes expressed in the developing urinary tract (WDPCP, OTX1, BMP5, VANGL1, and WNT5A). In particular, 3.3% of VUR patients were homozygous for an intronic variant in WDPCP (rs13013890; OR, 3.65; 95% CI, 2.39 to 5.56; P=1.86×10-9). This locus was associated with multiple genitourinary phenotypes in the UK Biobank and eMERGE studies. Analysis of Wnt5a mutant mice confirmed the role of Wnt5a signaling in bladder and ureteric morphogenesis. CONCLUSIONS: These data demonstrate the genetic heterogeneity of VUR. Altogether, 6% of patients with VUR harbored a rare CNV or a common variant genotype conferring an OR >3. Identification of these genetic risk factors has multiple implications for clinical care and for analysis of outcomes in VUR.

Incidence of Fragile X syndrome in Ireland.

Described as the commonest single gene cause of learning disability internationally, the incidence of Fragile X syndrome (FXS) has never previously been determined in Ireland. The aim of this work was to determine the observed incidence of FXS in the island of Ireland; the Republic of Ireland (ROI) and Northern Ireland (NI) separately and combined. Ascertainment was achieved for a cross-sectional study by a retrospective, clinical and laboratory database review of positive FXS cases, born in either ROI or NI, between years 2000-2009 inclusive. The observed incidence of FXS per 10,000 live births in the island of Ireland in males was 0.94 (95%CI: 0.75-1.13) or ∼1:10,600 and in females was 0.23 (95%CI: 0.14-0.32) or ∼1:43,000. Comparable testing rates for FXS are present in ROI and NI, with on average 1.48% (1.30% in ROI, 1.96% in NI) of live male births and 0.4% (0.35% in ROI, 0.55% in NI) of live female births undergoing analysis which is comparable to other centres internationally. This study demonstrates the observed incidence of FXS in the island of Ireland is (i) approximately half the estimated worldwide incidence in males and is not explained by low levels of testing, and (ii) approximately one quarter the estimated worldwide incidence in females which may be explained by low levels of testing. © 2017 Wiley Periodicals, Inc.

Urinary tract effects of HPSE2 mutations.

Urofacial syndrome (UFS) is an autosomal recessive congenital disease featuring grimacing and incomplete bladder emptying. Mutations of HPSE2, encoding heparanase 2, a heparanase 1 inhibitor, occur in UFS, but knowledge about the HPSE2 mutation spectrum is limited. Here, seven UFS kindreds with HPSE2 mutations are presented, including one with deleted asparagine 254, suggesting a role for this amino acid, which is conserved in vertebrate orthologs. HPSE2 mutations were absent in 23 non-neurogenic neurogenic bladder probands and, of 439 families with nonsyndromic vesicoureteric reflux, only one carried a putative pathogenic HPSE2 variant. Homozygous Hpse2 mutant mouse bladders contained urine more often than did wild-type organs, phenocopying human UFS. Pelvic ganglia neural cell bodies contained heparanase 1, heparanase 2, and leucine-rich repeats and immunoglobulin-like domains-2 (LRIG2), which is mutated in certain UFS families. In conclusion, heparanase 2 is an autonomic neural protein implicated in bladder emptying, but HPSE2 variants are uncommon in urinary diseases resembling UFS.

Heterozygous non-synonymous ROBO2 variants are unlikely to be sufficient to cause familial vesicoureteric reflux.

ROBO2, the receptor of SLIT2, is one of many genes/proteins that regulate the outgrowth of the ureteric bud, which is the first step in the development of the metanephric urinary system. Non-synonymous variants in ROBO2 have been found in a small proportion of patients with primary vesicoureteric reflux (VUR) in various countries. Here we sequenced 1 kb of promoter and all exons of ROBO2b with intronic margins in 227 index cases with primary VUR in an Irish population and found 55 variants, of which 20 were novel. We assessed the variants for evolutionary conservation and investigated novel and uncommon known conserved variants in 23 further index cases and family members of all index cases (to check for segregation with VUR), and then in healthy controls if we found segregation of the variants with VUR. Apart from one non-synonymous variant that was previously found in controls, we did not find any of the six other previously reported non-synonymous variants, but found four new non-synonymous variants. Of those, only two segregated with the disorder (p.Pro522Thr and p.Val799Ile). The former was not present in any of 592 healthy controls; the latter was present in one control. There are now 35 reported non-synonymous coding variants of ROBO2b. The predicted pathogenicity of those that have so far been found exclusively in VUR patients does not differ from that predicted for those variants also found in controls. Thus, our finding does not completely rule out that some variants may be the sole cause of VUR, but it is clear from the overall frequency that most of them cannot be. However, it is possible that some of these variants may cause VUR in combination with a mutation in another gene.

The Single-Access Technique for Impella Protected Percutaneous Coronary Intervention: A Single-Center Experience.

BACKGROUND: In this era of complex high-risk percutaneous coronary intervention (HR-PCI), mechanical support is being increasingly used. Traditionally, mechanical circulatory support with the Impella CP device requires a large-bore arterial access and an additional arterial access for the subsequent coronary intervention. We present a case series of the novel single-access for HR-PCI (SHIP) technique, in which a single arterial access is used for both mechanical support and coronary intervention. We reviewed 35 patients from June 2019 to February 2021 in whom the procedure was successfully employed in all but 1 case. In our case series, this technique appeared to be safe and feasible, and none of our patients suffered any major bleeding or vascular complications. As our experience with the procedure grew, we started employing this technique in patients presenting with ST-segment elevation myocardial infarction and cardiogenic shock. We discuss in detail the nuances of the technique, including troubleshooting, procedural characteristics, and complications, and review the currently available literature.

Predictors of high-degree atrioventricular block in patients with new-onset left bundle branch block following transcatheter aortic valve replacement.

BACKGROUND: The development of new left bundle branch block (LBBB) is frequently seen post TAVR and is a known risk factor for progression to high degree AV block. The timing and likelihood of progression into complete heart block is variable and can develop after hospital discharge. We sought to determine predictors for the development of high degree AV block in patients who developed LBBB following TAVR. METHODS: All patients between 2014 and 2019 underwent electrophysiology study after developing LBBB post TAVR. Data on these patients including baseline characteristics, echo parameters, EKG variables, HV interval, and the need for subsequent pacemaker implantation were extracted. A prolonged HV interval was defined as ≥ 65 ms. Clinically significant conduction abnormality was defined as development of high-degree AV block or clinically significant complete heart block. RESULTS: Thirty-four patients were included in our study of which 10 (29.4%) developed clinically significant heart block, while 24 (70.6%) did not. The mean HV interval for patients with clinically significant heart block was 70.1 ms vs 57.8 ms for those who did not (p = 0.022). Pre-existing first-degree heart block prior to TAVR (p = 0.026), history of AFib (p = 0.05) in addition to STS score (p = 0.037) were predictors of development of high-degree AV block in our patient population. CONCLUSIONS: In patients who develop LBBB following TAVR, HV interval, pre-existing first-degree heart block, and STS score predict progression to high-degree AV block. Performance of a routine electrophysiology study should be considered for high-risk patients who develop LBBB following TAVR.

Myocardial infarction secondary to coronary embolus in a patient with left ventricular non-compaction cardiomyopathy: a case report.

BACKGROUND: Coronary embolism is a rare cause of myocardial infarction (MI). We present a case report which emphasizes the importance of intracoronary imaging in these cases to identify the pathophysiological mechanism of MI. CASE SUMMARY: A 53-year-old male with no past medical history presented to the hospital with typical angina. Electrocardiogram and serum troponin I level trend confirmed non-ST-elevation myocardial infarction. Coronary angiography showed no evidence of any obstructive coronary artery disease, but two small thrombi were noted in the distal first obtuse marginal branch. Optical coherence tomography imaging confirmed this finding in absence of any underlying atherosclerotic plaque rupture or erosion. Cardiac magnetic resonance imaging revealed the diagnosis of non-compaction cardiomyopathy with severely depressed left ventricular function. Transmural MI was revealed by late gadolinium enhancement in the mid-lateral wall. Based on the pathophysiology of the MI confirmed by intracoronary imaging, antiplatelet medications were discontinued, and the patient was discharged on warfarin. Medical therapy was initiated for his cardiomyopathy. The patient recovered well and was asymptomatic at 1-year follow-up visit. DISCUSSION: Intracoronary imaging plays an important role to supplement coronary angiography to confirm the pathophysiology of MI in coronary embolism cases. This is important as it alters management in these patients.

Investigation of DNA variants specific to ROBO2 Isoform 'a' in Irish vesicoureteric reflux patients reveals marked CpG island variation.

ROBO2 gene disruption causes vesicoureteric reflux (VUR) amongst other congenital anomalies. Several VUR patient cohorts have been screened for variants in the ubiquitously expressed transcript, ROBO2b, but, apart from low levels in a few adult tissues, ROBO2a expression is confined to the embryo, and might be more relevant to VUR, a developmental disorder. ROBO2a has an alternative promoter and two alternative exons which replace the first exon of ROBO2b. We screened probands from 251 Irish VUR families for DNA variants in these. The CpG island of ROBO2a, which includes the non-coding first exon, was found to contain a run of six variants abolishing/creating CpG dinucleotides, including a novel variant, present in the VUR cases in one family, that was not present in 592 healthy Irish controls. In three of these positions, the CpG was created by the non-reference allele, and the reference allele was not the nucleotide that would result from spontaneous deamination of methylcytosine to thymine, suggesting that there might have been selection for variability in number of CpGs in this island. This is in marked contrast to the CpG island at the start of ROBO2b, which only contained a single variant that abolishes a CpG.

National Newborn Screening for cystic fibrosis in the Republic of Ireland: genetic data from the first 6.5 years.

Cystic fibrosis (CF) is the most common life-limiting autosomal recessive disease in the Republic of Ireland (ROI), with a previously quoted incidence of 1 in 1353 and carrier rate of 1 in 19. The National Newborn Screening (NBS) for CF was incorporated in July 2011 in the ROI. A cut-off point of the top 1% Immunoreactive Trypsinogen (IRT) was taken as an indication for 38 CFTR variant panel to maximise identification of affected CF cases and to minimise detection of carriers. All neonates from July 2011 to Dec 2017 with an elevated IRT on NBS were tested with 38 CFTR mutation panel and included. Clinical and laboratory database were analysed. In the first 6.5 years a total of 5,053 newborns (1.16% of total births) were screened with 38 CFTR panel. 170 CF affected cases, 320 unaffected carriers, 32 CF Screening Positive Inconclusive Diagnosis (CFSPID) were identified. There was one missed diagnosis. The most common disease-causing variant was c.1521_1523delCTT (p.(Phe508del)) followed by c.1652G>A (p.(Gly551Asp)). 95 out of 170 (55%) affected newborns were homozygous for c.1521_1523delCTT (p.(Phe08del)) and 25 (15%) carried at least one copy of c.1652G>A (p.(Gly551Asp)). Hence, 70% of affected newborns were eligible for CFTR modulator treatment. The NBS programme has identified almost triple the number of affected newborn with c.1652G>A (p.(Gly551Asp)) than previously quoted figures and identified less than 50% of carriers than predicted. The revised incidence and carrier frequency of CF in the ROI is 1 in 2570 and 1 in 25, respectively.

The increased incidence of the RET p.Gly691Ser variant in French-Canadian vesicoureteric reflux patients is not replicated by a larger study in Ireland.

The p.Gly691Ser variant of the RET protein, resulting from the 'A' allele of the SNP rs1799939 in exon 11 of the RET gene, was recently found to be present in a high proportion of primary vesicoureteric reflux (pVUR) patients in Quebec. We have determined the genotype of this SNP in 221 unrelated index cases of pVUR from the Irish population, in 190 full siblings of 160 of the index cases, and in 592 healthy controls. We found no significant difference in genotype or allele frequencies in patients and controls, and no tendency of affected siblings to share the same genotype. We also found no difference in the presence of additional phenotypic features such as duplex kidneys, between patients with and without the 'A' allele, and no difference in grade of reflux. We find no evidence of any influence of RET SNP rs1799939 on pVUR phenotype.

Managing uncertainty in inherited cardiac pathologies-an international multidisciplinary survey.

Multi-gene testing is useful in genetically heterogeneous conditions, including inherited cardiac pathologies. Increasing the number of genes analysed increases diagnostic yield of variants of certain, likely, or uncertain pathogenicity. Concerns exist regarding management of variants of uncertain/likely pathogenicity in conditions of oligogenic inheritance or variable expressivity. We surveyed a sample of colleagues across different specialties and departments internationally to compare management of patients with class 3 or class 4 variants in genes associated with non-syndromic cardiomyopathy or arrhythmia. An electronic survey regarding clinical management of variants ( www.surveymonkey.com/r/cardiacvariants ) was designed and distributed to colleagues internationally via professional bodies and direct email. 150 respondents (88 centres, 27 countries) completed the survey, most of whom were Clinical Geneticists or Genetic Counsellors. Most respondents offer pre-symptomatic testing to asymptomatic relatives of an individual with class 4 or class 5 variants. A minority of respondents offer pre-symptomatic testing for class 3 variants. Considering class 4 variants, 22 (15%) are fully reassuring that the patient with a negative predictive test would not develop the familial phenotype, while 123 (82%) counselled patients about the possibility of variant reclassification. Variability existed between and within centres and specialties. Multiple "free text" comments were provided. Recurring themes including need for multidisciplinary input, technical concerns, and concern regarding duty to review variants of uncertain significance. This study demonstrates that variability in management of likely pathogenic/uncertain variants exists. Close multi-disciplinary input is essential. The development of disorder or gene-specific evidence-based guidelines might ameliorate uncertainty in management.

Evaluation and use of a synthetic quality control material, included in the European external quality assessment scheme for cystic fibrosis.

Assuring high quality within the field of genetic testing is fundamental, as the results can have considerable impact on the patient and his or her family. The use of appropriate quality control (QC) samples is therefore essential. Diagnostic laboratories mainly use patient samples as QC material, which of course include a maximum of two mutations per sample. Bearing in mind that some assays (such as for cystic fibrosis [CF] testing) can test for more than 100 mutations, multiplex QC materials including more than two mutations could save valuable time and reagents. Based on this need, synthetic multiplex controls have been developed by Maine Molecular Quality Controls, Inc. (MMQCI) for CF. A synthetic control, containing six homozygous mutations and one polymorphism for CF transmembrane conductance regulator (CFTR), was evaluated by distributing it through the CF external quality assessment (EQA) scheme, along with the EQA samples in 2005. A total of 197 participants returned results of the yearly EQA scheme and 133 laboratories participated in the evaluation of the synthetic sample. Respectively, 76% and 73% of the participants were assigned as successful. This evaluation study revealed that the multiplex QC material performed well in the majority of assays and could be useful in method validation, as a tool to challenge interpretation skills, and as potential proficiency testing (PT) material.

Publisher Correction: Genome-wide linkage and association study implicates the 10q26 region as a major genetic contributor to primary nonsyndromic vesicoureteric reflux.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Genome-wide linkage and association study implicates the 10q26 region as a major genetic contributor to primary nonsyndromic vesicoureteric reflux.

Vesicoureteric reflux (VUR) is the commonest urological anomaly in children. Despite treatment improvements, associated renal lesions - congenital dysplasia, acquired scarring or both - are a common cause of childhood hypertension and renal failure. Primary VUR is familial, with transmission rate and sibling risk both approaching 50%, and appears highly genetically heterogeneous. It is often associated with other developmental anomalies of the urinary tract, emphasising its etiology as a disorder of urogenital tract development. We conducted a genome-wide linkage and association study in three European populations to search for loci predisposing to VUR. Family-based association analysis of 1098 parent-affected-child trios and case/control association analysis of 1147 cases and 3789 controls did not reveal any compelling associations, but parametric linkage analysis of 460 families (1062 affected individuals) under a dominant model identified a single region, on 10q26, that showed strong linkage (HLOD = 4.90; ZLRLOD = 4.39) to VUR. The ~9Mb region contains 69 genes, including some good biological candidates. Resequencing this region in selected individuals did not clearly implicate any gene but FOXI2, FANK1 and GLRX3 remain candidates for further investigation. This, the largest genetic study of VUR to date, highlights the 10q26 region as a major genetic contributor to VUR in European populations.

Best practice guidelines for the molecular genetic diagnosis of Type 1 (HFE-related) hereditary haemochromatosis.

BACKGROUND: Hereditary haemochromatosis (HH) is a recessively-inherited disorder of iron over-absorption prevalent in Caucasian populations. Affected individuals for Type 1 HH are usually either homozygous for a cysteine to tyrosine amino acid substitution at position 282 (C282Y) of the HFE gene, or compound heterozygotes for C282Y and for a histidine to aspartic acid change at position 63 (H63D). Molecular genetic testing for these two mutations has become widespread in recent years. With diverse testing methods and reporting practices in use, there was a clear need for agreed guidelines for haemochromatosis genetic testing. The UK Clinical Molecular Genetics Society has elaborated a consensus process for the development of disease-specific best practice guidelines for genetic testing. METHODS: A survey of current practice in the molecular diagnosis of haemochromatosis was conducted. Based on the results of this survey, draft guidelines were prepared using the template developed by UK Clinical Molecular Genetics Society. A workshop was held to develop the draft into a consensus document. The consensus document was then posted on the Clinical Molecular Genetics Society website for broader consultation and amendment. RESULTS: Consensus or near-consensus was achieved on all points in the draft guidelines. The consensus and consultation processes worked well, and outstanding issues were documented in an appendix to the guidelines. CONCLUSION: An agreed set of best practice guidelines were developed for diagnostic, predictive and carrier testing for hereditary haemochromatosis and for reporting the results of such testing.

Uroplakin III is not a major candidate gene for primary vesicoureteral reflux.

Vesicoureteral reflux (VUR) is the retrograde flow of urine from the bladder into the ureter and towards the kidneys. VUR is the most common cause of end stage renal failure in both children and adults and it is a major cause of severe hypertension in children. VUR is seen in approximately 1-2% of newborn Caucasians. Substantial evidence exists that VUR is a genetic disorder. Uroplakins are integral membrane proteins found in the bladder wall. Knockout studies in mice have suggested uroplakin III (UPK3) as a candidate gene for VUR. We have used parametric and nonparametric linkage analysis and tests for association, to investigate this possibility in a cohort of 126 sibling pairs affected with primary VUR. None of the analyses showed any substantial evidence for linkage or association of markers at the UPK3 locus to VUR. Our results do not support a role for UPK3 in primary VUR.

Comparison of the CFTR mutation spectrum in three cohorts of patients of Celtic origin from Brittany (France) and Ireland.

This study aims to compare the spectrum of the mutations identified in the gene responsible for cystic fibrosis in three cohorts of patients of Celtic origin from Brittany and Ireland. It included 389 patients from Brittany, 631 from Dublin and 139 from Cork. The CFTR gene analysis relied on the detection of the most common mutations, followed by a complete gene scanning using DGGE or D-HPLC. High mutation detection rates were obtained in each cohort: 99.6%, 96.8%, and 96.0% respectively. A high frequency of the c.1652_1655 del3 mutation (F508del: 74.8% to 81.3%) and of the "Celtic" mutation (c.1784G>A (G551D): 3.7% to 9.7%) was observed in each population. Apart from this, the mutation spectrums differed. In Brittany, the most common abnormalities were: c.1078delT (3.6%), c.4041C>G (N1303K: 1.4%), c.2670G>A (W846X(2): 1.0%) and c.1717-1G>A (1.0%), whereas in the cohort of Dublin, the main mutations were: c.482G>A (R117H: 3.0%), c.1811G>C (R560T: 2.4%) and c.621+1G>T (1.7%). Finally, in the Cork area, only the c.482G>A mutation (R117H) reached a frequency of 1%. Two previously-unreported mutations were identified in the Dublin cohort: c.2623-2A>G and c.3446T>G (M1105R). This collaborative study highlights the similarities of the CFTR alleles in the Breton and Irish populations, but also the disparities that exist between these populations, despite their common origin. Each population has its own history, with its mixture of founder effects and genetic drifts, which are at the origin of the current mutation distribution. The molecular study of the CFTR gene provides new tools for retracing European populations' histories.

Intrafamilial phenotypic variability in Friedreich ataxia associated with a G130V mutation in the FRDA gene.

BACKGROUND: Most patients with Friedreich ataxia (FA) have a GAA trinucleotide repeat expansion in intron 1 of the FA gene (FRDA) on both arms of chromosome 9. However, some patients are compound heterozygotes and harbor a GAA expansion on one allele and a point mutation on the other. Compound heterozygous patients with FA who have a GAA expansion and a G130V mutation have been reported to have an atypical phenotype with a slow disease progression, minimal or no ataxia, or gait spasticity. OBJECTIVE: To describe intrafamilial phenotypic variability in a GAA expansion/G130V mutation compound heterozygous family with FA. SETTING: Tertiary referral university hospital setting. PATIENTS AND METHODS: A 34-year-old man presented to our hospital with a 24-year history of stiff legs and mild unsteadiness of gait. Clinical examination showed a spastic paraparesis with normal to pathologically brisk deep tendon reflexes and mild left upper limb ataxia. His 27-year-old sister presented with a slowly progressive early-onset ataxic syndrome. She had ataxia of gait, mild to severe limb ataxia, and reduced or absent deep tendon reflexes, but no evidence of spasticity on examination. RESULTS: Neurophysiologic investigations showed evidence of a sensory axonal neuropathy, and molecular genetic analysis showed that both siblings were compound heterozygotes with a GAA expansion and a G130V mutation. CONCLUSIONS: This report confirms that compound heterozygous patients with FA who have a GAA expansion and a G130V mutation may present with an ataxic phenotype and that intrafamilial phenotypic variability in these pedigrees can occur. It also emphasizes the importance of performing molecular genetic analysis for the GAA trinucleotide expansion in patients presenting with a spastic paraparesis of undetermined etiology, especially when there is neurophysiologic evidence of a sensory axonal neuropathy.

Recommendations for reporting results of diagnostic genetic testing (biochemical, cytogenetic and molecular genetic).

Genetic test results can have considerable importance for patients, their parents and more remote family members. Clinical therapy and surveillance, reproductive decisions and genetic diagnostics in family members, including prenatal diagnosis, are based on these results. The genetic test report should therefore provide a clear, concise, accurate, fully interpretative and authoritative answer to the clinical question. The need for harmonizing reporting practice of genetic tests has been recognised by the External Quality Assessment (EQA), providers and laboratories. The ESHG Genetic Services Quality Committee has produced reporting guidelines for the genetic disciplines (biochemical, cytogenetic and molecular genetic). These guidelines give assistance on report content, including the interpretation of results. Selected examples of genetic test reports for all three disciplines are provided in an annexe.