Noninvasive prenatal diagnosis of Mendelian disorders for consanguineous couples by relative genotype dosage.

Noninvasive prenatal diagnosis relies on the presence in maternal blood of circulating cell-free fetal DNA released by apoptotic trophoblast cells. Widely used for aneuploidy screening, it can also be applied to monogenic diseases (NIPD-M) in case of known parental mutations. Due to the confounding effect of maternal DNA, detection of maternal or biparental mutations requires relative haplotype dosage (RHDO), a method relying on the presence of SNPs that are heterozygous in one parent and homozygous in the other. Unavoidably, there is a risk of test failure by lack of such informative SNPs, an event particularly likely for consanguineous couples who often share common haplotypes in regions of identity-by-descent. Here we present a novel approach, relative genotype dosage (RGDO) that bypasses this predicament by directly assessing fetal genotype with SNPs that are heterozygous in both parents (frequent in regions of identity-by-descent). We show that RGDO is as sensitive as RHDO and that it performs well over a large range of fetal fractions and DNA amounts, thereby opening NIPD-M to most consanguineous couples. We also report examples of couples, consanguineous or not, where combining RGDO and RHDO allowed a diagnosis that would not have been possible with only one approach.

[Rare causes of Hypophosphatemia: diagnostic approach]. / Hypophosphatémies de causes rares : approche diagnostique.

Hypophosphatemia is common and may be overlooked due to its asymptomatic nature or non-specific symptoms. Two main mechanisms are at its origin: a shift towards the intracellular sector and an increase in urinary phosphate excretion. A measurement of the urinary phosphate reabsorption threshold allows a diagnostic orientation. Alongside common forms of parathyroid hormone-dependent hypophosphatemia, one should not ignore rare FGF23-mediated forms, in particular X-linked hypophosphatemic rickets. The treatment, above all etiological, also includes the administration of phosphate and, in the event of an excess of FGF23, supplementation with calcitriol. In cases of oncogenic osteomalacia and X-linked hypophosphatemic rickets, the use of burosumab, an anti-FGF23 antibody, must be considered.

L'hypophosphatémie est fréquente. Pourtant, elle peut parfois être méconnue de par son caractère asymptomatique ou ses symptômes non spécifiques. Deux grands mécanismes sont à son origine : un shift vers le secteur intracellulaire et une augmentation de l'excrétion urinaire de phosphate. Une mesure du seuil de réabsorption urinaire de phosphate permet une orientation diagnostique. À côté de formes communes d'hypophosphatémies parathormone-dépendantes, il ne faut pas méconnaître des formes rares FGF23 médiées, en particulier le rachitisme hypophosphatémique lié à l'X. Le traitement, avant tout étiologique comporte aussi l'administration de phosphate et lors d'un excès de FGF23, une supplémentation en calcitriol. En cas d'ostéomalacie oncogénique et de rachitisme hypophosphatémique lié à l'X, l'emploi de burosumab, anticorps anti-FGF23, doit être considéré.

Bi-allelic loss of ERGIC1 causes relatively mild arthrogryposis.

Arthrogryposis describes the presence of multiple joint-contractures. Clinical severity of this phenotype is variable, and more than 400 causative genes have been proposed. Among these, ERGIC1 is a recently reported candidate encoding a putative transmembrane protein of the ER-Golgi interface. Two homozygous missense variants have been reported in patients with relatively mild non-syndromic arthrogryposis. In a consanguineous family with two affected siblings presenting congenital arthrogryposis and some facial dysmorphism we performed prenatal array-CGH, postnatal targeted exome and genome sequencing. Genome sequencing identified a homozygous 22.6 Kb deletion encompassing the promoter and first exon of ERGIC1. mRNA quantification showed the complete absence of ERGIC1 expression in the two affected siblings and a decrease in heterozygous parents. Our observations validate the pathogenic role of ERGIC1 in congenital arthrogryposis and demonstrate that complete loss of function causes a relatively mild phenotype. These findings will contribute to improve genetic counseling of ERGIC1 mutations.

Complex movement disorder in a patient with heterozygous YY1 mutation (Gabriele-de Vries syndrome).

YY1 mutations cause Gabriele-de Vries syndrome, a recently described condition involving cognitive impairment, facial dysmorphism and intrauterine growth restriction. Movement disorders were reported in 5/10 cases of the original series, but no detailed description was provided. Here we present a 21-year-old woman with a mild intellectual deficit, facial dysmorphism and a complex movement disorder including an action tremor, cerebellar ataxia, dystonia, and partial ocular apraxia as the presenting and most striking feature. Whole-exome sequencing revealed a novel heterozygous de novo mutation in YY1 [NM: 003403.4 (YY1): c.907 T > C; p.(Cys303Arg)], classified as pathogenic according to the ACMG guidelines.

PBX1 haploinsufficiency leads to syndromic congenital anomalies of the kidney and urinary tract (CAKUT) in humans.

BACKGROUND: Congenital anomalies of the kidney and urinary tract (CAKUT) represent a significant healthcare burden since it is the primary cause of chronic kidney in children. CNVs represent a recurrent molecular cause of CAKUT but the culprit gene remains often elusive. Our study aimed to define the gene responsible for CAKUT in patients with an 1q23.3q24.1 microdeletion. METHODS: We describe eight patients presenting with CAKUT carrying an 1q23.3q24.1 microdeletion as identified by chromosomal microarray analysis (CMA). Clinical features were collected, especially the renal and urinary tract phenotype, and extrarenal features. We characterised PBX1 expression and localisation in fetal and adult kidneys using quantitative RT-PCR and immunohistochemistry. RESULTS: We defined a 276-kb minimal common region (MCR) that only overlaps with the PBX1 gene. All eight patients presented with syndromic CAKUT. CAKUT were mostly bilateral renal hypoplasia (75%). The most frequent extrarenal symptoms were developmental delay and ear malformations. We demonstrate that PBX1 is strongly expressed in fetal kidneys and brain and expression levels decreased in adult samples. In control fetal kidneys, PBX1 was localised in nuclei of medullary, interstitial and mesenchymal cells, whereas it was present in endothelial cells in adult kidneys. CONCLUSIONS: Our results indicate that PBX1 haploinsufficiency leads to syndromic CAKUT as supported by the Pbx1-null mice model. Correct PBX1 dosage appears to be critical for normal nephrogenesis and seems important for brain development in humans. CMA should be recommended in cases of fetal renal anomalies to improve genetic counselling and pregnancy management.

Defining categories of actionability for secondary findings in next-generation sequencing.

Next-generation sequencing is increasingly used in clinical practice for the diagnosis of Mendelian diseases. Because of the high likelihood of secondary findings associated with this technique, the process of informing patients is beset with new challenges. One of them is regarding the type of secondary findings that ought to be disclosed to patients. The aim of this research is to propose a practical implementation of the notion of actionability, a common criteria justifying the disclosure of secondary findings but whose interpretation varies greatly among professionals. We distinguish three types of actionability corresponding to (1) well-established medical actions, (2) patient-initiated health-related actions and (3) life-plan decisions. We argue that actionability depends on the characteristics of the mutation or gene and on the values of patients. In discussing the return of secondary findings, it is important that the physician tries to get an impression of the specific situation and values of patients. Regarding variants of uncertain clinical significance in actionable genes, we found that different understandings of autonomy lead to different conclusions and that, for some of them, it may be legitimate to refrain from returning uncertain information.

Severe and Progressive Fetal Ventriculomegaly Leading to the Diagnosis of Periventricular Nodular Heterotopias with Good Outcome.

Severe fetal ventriculomegaly is generally associated with poor prognosis in terms of survival and neurodevelopment outcome. As such, many parents opt to terminate the pregnancy independently of a known etiology. We report here the case of a female fetus with severe progressive ventriculomegaly due to the unexpected presence of bilateral nodular periventricular heterotopias visualized on MRI of a fetal brain. Reaching a structural diagnosis was perceived as a relief for the parents and the pregnancy was continued. Neurodevelopment assessment at 3 years of age is normal with no epilepsy.

Diagnostic exome sequencing to elucidate the genetic basis of likely recessive disorders in consanguineous families.

Rare, atypical, and undiagnosed autosomal-recessive disorders frequently occur in the offspring of consanguineous couples. Current routine diagnostic genetic tests fail to establish a diagnosis in many cases. We employed exome sequencing to identify the underlying molecular defects in patients with unresolved but putatively autosomal-recessive disorders in consanguineous families and postulated that the pathogenic variants would reside within homozygous regions. Fifty consanguineous families participated in the study, with a wide spectrum of clinical phenotypes suggestive of autosomal-recessive inheritance, but with no definitive molecular diagnosis. DNA samples from the patient(s), unaffected sibling(s), and the parents were genotyped with a 720K SNP array. Exome sequencing and array CGH (comparative genomic hybridization) were then performed on one affected individual per family. High-confidence pathogenic variants were found in homozygosity in known disease-causing genes in 18 families (36%) (one by array CGH and 17 by exome sequencing), accounting for the clinical phenotype in whole or in part. In the remainder of the families, no causative variant in a known pathogenic gene was identified. Our study shows that exome sequencing, in addition to being a powerful diagnostic tool, promises to rapidly expand our knowledge of rare genetic Mendelian disorders and can be used to establish more detailed causative links between mutant genotypes and clinical phenotypes.

Chromosomal rearrangements in patients with clinical features of Silver-Russell syndrome.

Silver-Russell syndrome (SRS) is characterized by pre- and postnatal growth retardation, relative macrocephaly, asymmetry, and a triangular facial gestalt. In 5-10% of the patients the phenotype is caused by maternal UPD 7, and 38-64% of the patients present with hypomethylation at the imprinting center region 1 (ICR1) on 11p15.5. The etiology of the remaining cases is so far not known and various (sub-)microscopic chromosome aberrations with a phenotype resembling SRS have been published, especially duplication 11p15 (n = 15), deletion 12q14 (n = 19), ring chromosome 15, deletion 15qter, and various other mostly unique chromosomal aberrations (n = 30). In this study the phenotypes of these chromosomal aberrations were revisited and compared with the phenotypes of maternal UPD 7 and hypomethylation at ICR1 on 11p15.5. In some patients with a unique chromosomal aberration even the hallmarks of SRS were missing. Patients with duplication 11p15 show a more variable occipitofrontal head circumference at birth, a higher frequency of intellectual disability, and additional anomalies not reported in SRS. Deletion 12q14 is characterized by less severe pre- and postnatal growth retardation and less impressive relative macrocephaly. Patients with ring chromosome 15 and deletion 15qter have no relative macrocephaly (mostly even microcephaly) and more severe intellectual disability. Finally, deletion 15qter lacks the triangular facial gestalt. In summary, as SRS seems not an adequate diagnosis in many of these patients, diagnosis should focus on the chromosomal aberration than on SRS.

Garrod's fourth inborn error of metabolism solved by the identification of mutations causing pentosuria.

Pentosuria is one of four conditions hypothesized by Archibald Garrod in 1908 to be inborn errors of metabolism. Mutations responsible for the other three conditions (albinism, alkaptonuria, and cystinuria) have been identified, but the mutations responsible for pentosuria remained unknown. Pentosuria, which affects almost exclusively individuals of Ashkenazi Jewish ancestry, is characterized by high levels of the pentose sugar L-xylulose in blood and urine and deficiency of the enzyme L-xylulose reductase. The condition is autosomal-recessive and completely clinically benign, but in the early and mid-20th century attracted attention because it was often confused with diabetes mellitus and inappropriately treated with insulin. Persons with pentosuria were identified from records of Margaret Lasker, who studied the condition in the 1930s to 1960s. In the DCXR gene encoding L-xylulose reductase, we identified two mutations, DCXR c.583ΔC and DCXR c.52(+1)G > A, each predicted to lead to loss of enzyme activity. Of nine unrelated living pentosuric subjects, six were homozygous for DCXR c.583ΔC, one was homozygous for DCXR c.52(+1)G > A, and two were compound heterozygous for the two mutant alleles. L-xylulose reductase was not detectable in protein lysates from subjects' cells and high levels of xylulose were detected in their sera, confirming the relationship between the DCXR genotypes and the pentosuric phenotype. The combined frequency of the two mutant DCXR alleles in 1,067 Ashkenazi Jewish controls was 0.0173, suggesting a pentosuria frequency of approximately one in 3,300 in this population. Haplotype analysis indicated that the DCXR c.52(+1)G > A mutation arose more recently than the DCXR c.583ΔC mutation.

[Antenatal diagnosis: the revolution of new technologies]. / Gynécologie-obstétrique. Diagnostic prenatal: la révolution des nouvelles technologies.

Since ten years, the number of amniocenteses or chorionic villous sampling for maternal anxiety has decreased thanks to the first trimester screening of trisomy 21 by ultrasound and maternal serum analysis. Two new tools have recently revolutionized antenatal screening and diagnosis: Analysing fetal DNA in maternal blood for chromosomes 21, 18 and 13 in order to avoid invasive fetal sampling and genomic comparative hybridization in order to diagnose deletions or duplications not detected by conventional caryotyping. These new technologies are dedicated to high-risk pregnancies, and have limitations. They do not replace ultrasound or first trimester screening. Information and ethics are central in antenatal screening and diagnosis.

Case report: desmoplakin cardiomyopathy presenting as an inflammatory cardiomyopathy with repeated sudden cardiac arrests.

Background: Desmoplakin cardiomyopathy has been recently classified as a non-dilated left ventricular cardiomyopathy, which is characterized by inflammatory-like episodes followed by left ventricular fibrosis/dysfunction and ventricular arrhythmias. Specific management is unclear. Case summary: We report a detailed case of a 46-year-old Caucasian woman presenting with repeated sudden cardiac arrests who was diagnosed with a new variant in the desmoplakin gene. Because the initial 18F-fluorodeoxyglucose positron emission tomography scan showed significant hypermetabolism, she was treated with immunosuppressors, with only minimal improvement on imaging. Discussion: Desmoplakin cardiomyopathy should be considered in the differential diagnosis of inflammatory cardiomyopathies. Little is known about the use of immunosuppressive treatments, but it could be reasonable for some selected patients.

Odyssey of a Misclassified Genomic Variant: Insight from an Incidental Finding Assessment.

Genetic evaluation of a teenager with seizure found no pathogenic variant in a large gene panel, but an incidental likely pathogenic HNF4A variant, deemed to cause MODY1 diabetes. Diabetes history was absent and glycated hemoglobin normal, but serum calcium was severely low, with abnormally high parathyroid hormone. Thus, pseudohypoparathyroidism was suspected and confirmed by molecular genetic testing. Calcium and calcitriol supplementation led to calcium normalization and neurological symptom improvement. Given the absence of personal or family diabetes history, the HNF4A variant was reassessed and found to encode an alternative transcript with poor expression and activity levels, hence downgraded on expert advice from 'likely pathogenic' to 'likely benign'. Besides illustrating the importance of structured medical workup before launching extensive targeted exome sequencing, this case highlights the need for caution in incidental finding interpretation in patients lacking compatible phenotype or family history, and the value of expert advice in such variant interpretation.

Evolution and triggers of defibrillator shocks in patients with arrhythmogenic right ventricular cardiomyopathy.

INTRODUCTION: Implantable cardioverter-defibrillators (ICDs) can prevent sudden cardiac death due to ventricular arrhythmias in patients with arrhythmogenic right ventricular cardiomyopathy (ARVC). The aim of our study was to assess the cumulative burden, evolution and potential triggers of appropriate ICD shocks during long-term follow-up, which may help to reduce and further refine individual arrhythmic risk in this challenging disease. METHODS: This retrospective cohort study included 53 patients with definite ARVC according to the 2010 Task Force Criteria from the multicentre Swiss ARVC Registry with an implanted ICD for primary or secondary prevention. Follow-up was conducted by assessing all available patient records from patient visits, hospitalisations, blood samples, genetic analysis, as well as device interrogation and tracings. RESULTS: Fifty-three patients (male 71.7%, mean age 43±2.2 years, genotype positive 58.5%) were analysed during a median follow-up of 7.9 (IQR 10) years. In 29 (54.7%) patients, 177 appropriate ICD shocks associated with 71 shock episodes occurred. Median time to first appropriate ICD shock was 2.8 (IQR 3.6) years. Long-term risk of shocks remained high throughout long-term follow-up. Shock episodes occurred mainly during daytime (91.5%, n=65) and without seasonal preference. We identified potentially reversible triggers in 56 of 71 (78.9%) appropriate shock episodes, the main triggers representing physical activity, inflammation and hypokalaemia. CONCLUSION: The long-term risk of appropriate ICD shocks in patients with ARVC remains high during long-term follow-up. Ventricular arrhythmias occur more often during daytime, without seasonal preference. Reversible triggers are frequent with the most common triggers for appropriate ICD shocks being physical activity, inflammation and hypokalaemia in this patient population.

Rapid detection of genetic variants in hypertrophic cardiomyopathy by custom DNA resequencing array in clinical practice.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease (1/500) and the most common cause of sudden cardiac death in young people. Pathogenic mutation detection of HCM is having a growing impact on the medical management of patients and their families. However, the remarkable genetic and allelic heterogeneity makes molecular analysis by conventional methods very time-consuming, expensive and difficult to realise in a routine diagnostic molecular laboratory. METHOD AND RESULTS: The authors used their custom DNA resequencing array which interrogates all possible single-nucleotide variants on both strands of all exons (n=160), splice sites and 5'-untranslated region of 12 HCM genes (27 000 nucleotides). The results for 122 unrelated patients with HCM are presented. Thirty-three known or novel potentially pathogenic heterozygous single-nucleotide variants were identified in 38 patients (31%) in genes MYH7, MYBPC3, TNNT2, TNNI3, TPM1, MYL3 and ACTC1. CONCLUSIONS: Although next-generation sequencing will replace all large-scale sequencing platforms for inherited cardiac disorders in the near future, this HCM resequencing array is currently the most rapid, cost-effective and reasonably efficient technology for first-tier mutation screening of HCM in clinical practice. Because of its design, the array is also an appropriate tool for initial screening of other inherited forms of cardiomyopathy.

Brugada Syndrome Associated with Different Heterozygous SCN5A Variants in Two Unrelated Families.

The cardiac sodium channel (Nav1.5) controls cardiac excitability by triggering the action potential of cardiac myocytes and controlling electric impulse transmission. However, it has also been associated with arrhythmogenic cardiomyopathies. Accordingly, genetic variants in SCN5A that result in loss of function of Nav1.5 are associated with inherited arrhythmia syndromes, which are caused by reduced cardiac excitability, particularly Brugada syndrome (BrS) as well as arrhythmogenic right ventricular cardiomyopathy (ARVC). We report a novel pathogenic SCNA5 variant being associated with BrS overlapping with ARVC, as well as disease progression with a previously reported SCN5A variant being associated with a phenotype of BrS and conduction system disorder in two unrelated families.

Consanguineous marriages, pearls and perils: Geneva International Consanguinity Workshop Report.

Approximately 1.1 billion people currently live in countries where consanguineous marriages are customary, and among them one in every three marriages is between cousins. Opinions diverge between those warning of the possible health risks to offspring and others who highlight the social benefits of consanguineous marriages. A consanguinity study group of international experts and counselors met at the Geneva International Consanguinity Workshop from May 3, 2010, to May 7, 2010, to discuss the known and presumptive risks and benefits of close kin marriages and to identify important future areas for research on consanguinity. The group highlighted the importance of evidence-based counseling recommendations for consanguineous marriages and of undertaking both genomic and social research in defining the various influences and outcomes of consanguinity. Technological advances in rapid high-throughput genome sequencing and for the identification of copy number variants by comparative genomic hybridization offer a significant opportunity to identify genotype-phenotype correlations focusing on autozygosity, the hallmark of consanguinity. The ongoing strong preferential culture of close kin marriages in many societies, and among migrant communities in Western countries, merits an equivalently detailed assessment of the social and genetic benefits of consanguinity in future studies.

Quantitative sequence analysis of FBN1 premature termination codons provides evidence for incomplete NMD in leukocytes.

We improved, evaluated, and used Sanger sequencing for quantification of single nucleotide polymorphism (SNP) variants in transcripts and gDNA samples. This improved assay resulted in highly reproducible relative allele frequencies (e.g., for a heterozygous gDNA 50.0+/-1.4%, and for a missense mutation-bearing transcript 46.9+/-3.7%) with a lower detection limit of 3-9%. It provided excellent accuracy and linear correlation between expected and observed relative allele frequencies. This sequencing assay, which can also be used for the quantification of copy number variations (CNVs), methylations, mosaicisms, and DNA pools, enabled us to analyze transcripts of the FBN1 gene in fibroblasts and blood samples of patients with suspected Marfan syndrome not only qualitatively but also quantitatively. We report a total of 18 novel and 19 known FBN1 sequence variants leading to a premature termination codon (PTC), 26 of which we analyzed by quantitative sequencing both at gDNA and cDNA levels. The relative amounts of PTC-containing FBN1 transcripts in fresh and PAXgene-stabilized blood samples were significantly higher (33.0+/-3.9% to 80.0+/-7.2%) than those detected in affected fibroblasts with inhibition of nonsense-mediated mRNA decay (NMD) (11.0+/-2.1% to 25.0+/-1.8%), whereas in fibroblasts without NMD inhibition no mutant alleles could be detected. These results provide evidence for incomplete NMD in leukocytes and have particular importance for RNA-based analyses not only in FBN1 but also in other genes.

Management of delivery of a fetus with autosomal recessive polycystic kidney disease: a case report of abdominal dystocia and review of the literature.

BACKGROUND: Autosomal recessive renal polycystic kidney disease occurs in 1 in 20,000 live births. It is caused by mutations in both alleles of the PKHD1 gene. Management of delivery in cases of suspected autosomal recessive renal polycystic kidney disease is rarely discussed, and literature concerning abdominal dystocia is extremely scarce. We present a case of a patient with autosomal recessive renal polycystic kidney disease whose delivery was complicated by abdominal dystocia, and we discuss the factors that determined the route and timing of delivery. CASE PRESENTATION: A 23-year-old Caucasian woman, G2 P0, with a prior unremarkable pregnancy was referred to our tertiary center at 31 weeks of gestation because of severe oligoamnios (amniotic fluid index = 2) and hyperechogenic, dedifferentiated, and enlarged fetal kidneys. She had no other genitourinary anomaly. Fetal magnetic resonance imaging showed enlarged, hypersignal kidneys and severe pulmonary hypoplasia. We had a high suspicion of autosomal recessive renal polycystic kidney disease, and after discussion with our multidisciplinary team, the parents opted for conservative care. Ultrasound performed at 35 weeks of gestation showed a fetal estimated weight of 3550 g and an abdominal circumference of 377 mm, both above the 90th percentile. Because of the very rapid kidney growth and suspected risk of abdominal dystocia, we proposed induction of labor at 36 weeks of gestation after corticosteroid administration for fetal lung maturation. Vaginal delivery was complicated by abdominal dystocia, which resolved by continuing expulsive efforts and gentle fetal traction. A 3300-g (P50-90) male infant was born with Apgar scores of 1-7-7 at 1, 5, and 10 minutes, respectively, and arterial and venous umbilical cord pH values of 7.23-7.33. Continuous peritoneal dialysis was started at day 2 of life because of anuria. Currently, the infant is 1 year old and is waiting for kidney transplant that should be performed once he reaches 10 kg. Molecular analysis of PKHD1 performed on deoxyribonucleic acid (DNA) from the umbilical cord confirmed autosomal recessive renal polycystic kidney disease. CONCLUSIONS: Management of delivery in cases of suspected autosomal recessive renal polycystic kidney disease needs to be discussed because of the risk of abdominal dystocia. The route and timing of delivery depend on the size of the fetal abdominal circumference and the gestational age. The rate of kidney growth must also be taken into account.