Prenatal exome sequencing and chromosomal microarray analysis in fetal structural anomalies in a highly consanguineous population reveals a propensity of ciliopathy genes causing multisystem phenotypes.

Fetal abnormalities are detected in 3% of all pregnancies and are responsible for approximately 20% of all perinatal deaths. Chromosomal microarray analysis (CMA) and exome sequencing (ES) are widely used in prenatal settings for molecular genetic diagnostics with variable diagnostic yields. In this study, we aimed to determine the diagnostic yield of trio-ES in detecting the cause of fetal abnormalities within a highly consanguineous population. In families with a history of congenital anomalies, a total of 119 fetuses with structural anomalies were recruited and DNA from invasive samples were used together with parental DNA samples for trio-ES and CMA. Data were analysed to determine possible underlying genetic disorders associated with observed fetal phenotypes. The cohort had a known consanguinity of 81%. Trio-ES led to diagnostic molecular genetic findings in 59 fetuses (with pathogenic/likely pathogenic variants) most with multisystem or renal abnormalities. CMA detected chromosomal abnormalities compatible with the fetal phenotype in another 7 cases. Monogenic ciliopathy disorders with an autosomal recessive inheritance were the predominant cause of multisystem fetal anomalies (24/59 cases, 40.7%) with loss of function variants representing the vast majority of molecular genetic abnormalities. Heterozygous de novo pathogenic variants were found in four fetuses. A total of 23 novel variants predicted to be associated with the phenotype were detected. Prenatal trio-ES and CMA detected likely causative molecular genetic defects in a total of 55% of families with fetal anomalies confirming the diagnostic utility of trio-ES and CMA as first-line genetic test in the prenatal diagnosis of multisystem fetal anomalies including ciliopathy syndromes.

A null founder variant in NPNT, encoding nephronectin, causes autosomal recessive renal agenesis.

Congenital anomalies of the kidney and urinary tract (CAKUT) are a spectrum of abnormalities affecting morphogenesis of the kidneys and other structures of the urinary tract. Bilateral renal agenesis (BRA) is the most severe presentation of CAKUT. Loss of either nephronectin (NPNT) or its receptor ITGA8 leads to failure of metanephric kidney development with resulting renal agenesis in murine models. Very recently, a single family with renal agenesis and a homozygous truncating variant in NPNT was reported. We report two families in whom genome-wide linkage analysis showed an autozygous locus linked to BRA (at least one member has unilateral renal agenesis) at 4q24, with an LOD score of ~3. Exome sequencing detected a nonsense variant in NPNT in both families within the linkage interval. The pathogenicity of this variant was supported by reverse transcription polymerase chain reaction data showing complete nonsense-mediated decay of the NPNT transcript. Our report confirms the candidacy of NPNT in renal agenesis in humans and shows that even complete loss of function can be compatible with the formation of a single kidney.

Bialleleic PKD1 mutations underlie early-onset autosomal dominant polycystic kidney disease in Saudi Arabian families.

BACKGROUND: Polycystic kidney disease (PKD) is one of the most common genetic renal diseases and may be inherited in an autosomal dominant or autosomal recessive pattern. Pathogenic variants in two major genes, PKD1 and PKD2, and two rarer genes, GANAB and DNAJB11, cause autosomal dominant PKD (ADPKD). Early onset and severe PKD can occur with PKD1 and PKD2 pathogenic variants and such phenotypes may be modified by second alleles inherited in trans. Homozygous or compound heterozygous hypomorphic PKD1 variants may also cause a moderate to severe disease PKD phenotype. METHODS: Targeted renal gene panel followed by Sanger sequencing of PKD1 gene were employed to investigate molecular causes in early onset PKD patients. RESULTS: In this study, we report four consanguineous Saudi Arabian families with early onset PKD which were associated with biallelic variants in PKD1 gene. CONCLUSIONS: Our findings confirm that PKD1 alleles may combine to produce severe paediatric onset PKD mimicking the more severe autosomal recessive ciliopathy syndromes associated with PKD. Screening of parents of such children may also reveal subclinical PKD phenotypes.

Molecular autopsy in maternal-fetal medicine.

PurposeThe application of genomic sequencing to investigate unexplained death during early human development, a form of lethality likely enriched for severe Mendelian disorders, has been limited.MethodsIn this study, we employed exome sequencing as a molecular autopsy tool in a cohort of 44 families with at least one death or lethal fetal malformation at any stage of in utero development. Where no DNA was available from the fetus, we performed molecular autopsy by proxy, i.e., through parental testing.ResultsPathogenic or likely pathogenic variants were identified in 22 families (50%), and variants of unknown significance were identified in further 15 families (34%). These variants were in genes known to cause embryonic or perinatal lethality (ALPL, GUSB, SLC17A5, MRPS16, THSD1, PIEZO1, and CTSA), genes known to cause Mendelian phenotypes that do not typically include embryonic lethality (INVS, FKTN, MYBPC3, COL11A2, KRIT1, ASCC1, NEB, LZTR1, TTC21B, AGT, KLHL41, GFPT1, and WDR81) and genes with no established links to human disease that we propose as novel candidates supported by embryonic lethality of their orthologs or other lines of evidence (MS4A7, SERPINA11, FCRL4, MYBPHL, PRPF19, VPS13D, KIAA1109, MOCS3, SVOPL, FEN1, HSPB11, KIF19, and EXOC3L2).ConclusionOur results suggest that molecular autopsy in pregnancy losses is a practical and high-yield alternative to traditional autopsy, and an opportunity for bringing precision medicine to the clinical practice of perinatology.

Identification of a novel MKS locus defined by TMEM107 mutation.

Meckel-Gruber syndrome (MKS) is a perinatally lethal disorder characterized by the triad of occipital encephalocele, polydactyly and polycystic kidneys. Typical of other disorders related to defective primary cilium (ciliopathies), MKS is genetically heterogeneous with mutations in a dozen genes to date known to cause the disease. In an ongoing effort to characterize MKS clinically and genetically, we implemented a gene panel and next-generation sequencing approach to identify the causal mutation in 25 MKS families. Of the three families that did not harbor an identifiable causal mutation by this approach, two mapped to a novel disease locus in which whole-exome sequencing revealed the likely causal mutation as a homozygous splicing variant in TMEM107, which we confirm leads to aberrant splicing and nonsense-mediated decay. TMEM107 had been independently identified in two mouse models as a cilia-related protein and mutant mice display typical ciliopathy phenotypes. Our analysis of patient fibroblasts shows marked ciliogenesis defect with an accompanying perturbation of sonic hedgehog signaling, highly concordant with the cellular phenotype in Tmem107 mutants. This study shows that known MKS loci account for the overwhelming majority of MKS cases but additional loci exist including MKS13 caused by TMEM107 mutation.

Genetic spectrum of Saudi Arabian patients with antenatal cystic kidney disease and ciliopathy phenotypes using a targeted renal gene panel.

BACKGROUND: Inherited cystic kidney disorders are a common cause of end-stage renal disease. Over 50 ciliopathy genes, which encode proteins that influence the structure and function of the primary cilia, are implicated in cystic kidney disease. METHODS: To define the phenotype and genotype of cystic kidney disease in fetuses and neonates, we correlated antenatal ultrasound examination and postnatal renal ultrasound examination with targeted exon sequencing, using a renal gene panel. A cohort of 44 families in whom antenatal renal ultrasound scanning findings in affected cases included bilateral cystic kidney disease, echogenic kidneys or enlarged kidneys was investigated. RESULTS: In this cohort, disease phenotypes were severe with 36 cases of stillbirth or perinatal death. Extra renal malformations, including encephalocele, polydactyly and heart malformations, consistent with ciliopathy phenotypes, were frequently detected. Renal gene panel testing identified causative mutations in 21 out of 34 families (62%), where patient and parental DNA was available. In the remaining 10 families, where only parental DNA was available, 7 inferred causative mutations were found. Together, mutations were found in 12 different genes with a total of 13 novel pathogenic variants, including an inferred novel variant in NEK8. Mutations in CC2D2A were the most common cause of an antenatal cystic kidney disease and a suspected ciliopathy in our cohort. CONCLUSIONS: In families with ciliopathy phenotypes, mutational analysis using a targeted renal gene panel allows a rapid molecular diagnosis and provides important information for patients, parents and their physicians.

Missense Variants in GFRA1 and NPNT Are Associated with Congenital Anomalies of the Kidney and Urinary Tract.

The use of next-generation sequencing (NGS) has helped in identifying many genes that cause congenital anomalies of the kidney and urinary tract (CAKUT). Bilateral renal agenesis (BRA) is the most severe presentation of CAKUT, and its association with autosomal recessively inherited genes is expanding. Highly consanguineous populations can impact the detection of recessively inherited genes. Here, we report two families harboring homozygous missense variants in recently described genes, NPNT and GFRA1. Two consanguineous families with neonatal death due to CAKUT were investigated. Fetal ultrasound of probands identified BRA in the first family and severe renal cystic dysplasia in the second family. Exome sequencing coupled with homozygosity mapping was performed, and Sanger sequencing was used to confirm segregation of alleles in both families. In the first family with BRA, we identified a homozygous missense variant in GFRA1: c.362A>G; p.(Tyr121Cys), which is predicted to damage the protein structure. In the second family with renal cystic dysplasia, we identified a homozygous missense variant in NPNT: c.56C>G; p.(Ala19Gly), which is predicted to disrupt the signal peptide site. We report two Saudi Arabian consanguineous families with CAKUT phenotypes that included renal agenesis caused by missense variants in GFRA1 and NPNT, confirming the role of these two genes in human kidney development.

Maternal Vitamin D Levels and Its Correlation With Low Birth Weight in Neonates: A Tertiary Care Hospital Experience in Saudi Arabia.

Introduction A meta-analysis showed that 63.6% of the Saudi population have vitamin D deficiency, including many pregnant women. Studies showed that maternal vitamin D deficiency during pregnancy is a risk factor for low birth weight (LBW) in neonates. Neonatal LBW is a risk factor for multiple neonatal complications including respiratory distress syndrome, necrotizing enterocolitis, chronic renal disorders, seizures, and sepsis. Our objective in this study is to determine a correlation between low maternal vitamin D level and neonatal LBW in Saudi Arabia. Methods Neonates (n = 119) were divided based on their gestational age (GA) into full-term neonates (≥37 weeks) and preterm neonates (< 37 weeks) and based on birth weight into normal birth weight neonates (full-term = 2,500-3,500 g or preterm > 10th percentile) and LBW neonates (full-term < 2,500 g or preterm < 10th percentile). Vitamin D deficiency is defined as 25- hydroxyvitamin D level less than 50 nmol/L. Results Correlating neonatal birth weight with maternal vitamin D level during pregnancy was statistically insignificant for both full-term neonates and preterm neonates. In contrast, comparing the mean maternal vitamin D levels in each neonatal group showed that the mean were higher in mothers of neonates with normal birth weight. Conclusion Because 87.4% of mothers had low vitamin D levels during their pregnancy, correlation between maternal vitamin D level and LBW in neonates could not be found. However, mean maternal vitamin D levels were higher in mothers with normal birth weight neonates. Therefore, further detailed studies are required to establish local guidelines about the treatment of vitamin D deficiency during pregnancy.

Novel loss of function variants in FRAS1 AND FREM2 underlie renal agenesis in consanguineous families.

INTRODUCTION: Congenital anomalies of the kidney and urinary tract (CAKUT) are a group of abnormalities that affect structure of the kidneys or other structures of the urinary tract. The majority of CAKUT are asymptomatic and are diagnosed prenatally by ultrasound scanning or found incidentally in postnatal life. CAKUT varies in severity and may lead to life-threatening kidney failure and end-stage kidney disease. Renal agenesis, a severe form of CAKUT, is a congenital absence of one or both kidneys. Bilateral renal agenesis belongs to a group of prenatally lethal renal diseases and is often detected on fetal ultrasound scanning during the investigation of oligohydramnios. Approximately 40% of fetuses with bilateral renal agenesis are stillborn or die a few hours postnatally. Mutations in many renal development genes have been shown to be associated with renal agenesis. METHODS: Six consanguineous Saudi Arabian families were recruited to study the molecular genetic causes of recurrent miscarriages and lost fetuses due to oligohydramnios, renal agenesis and other congenital anomalies. Whole exome sequencing was employed to underlying detect genetic defects. RESULTS: Novel loss of function variants were detected in FRAS1 and FREM2. In FRAS1, a homozygous splice site variant c.9780+2T>C was found in an affected fetus, segregating form each parent. In addition, in three other families both parents were heterozygous for a frameshift variant (c.8981dupT; p.His2995Profs*3) and splice site variants (c.5217+1G>C and c.8098+2T>A), respectively. In FREM2, a homozygous nonsense variant (c.2303C>G; p.Ser768*) was found in an affected fetus, segregating from both parents. In another family, both parents carried a FREM2 heterozygous frameshift variant (c.3969delC; p.Asn1323Lysfs*5). CONCLUSION: We describe consanguineous families with clinical features of antenatal oligohydramnios and bilateral renal agenesis, in whom we have identified novel pathogenic variants in FRAS1 and FREM2. These finding highlights the association between mutations in FRAS1 and FREM2 and antenatal/perinatal death.

Fetal Anomalies Associated with Novel Pathogenic Variants in TMEM94.

BACKGROUND: Intellectual developmental disorder with cardiac defects and dysmorphic facies (IDDCDF, MIM 618316) is a newly described disorder. It is characterized by global developmental delay, intellectual disability and speech delay, congenital cardiac malformations, and dysmorphic facial features. Biallelic pathogenic variants of TMEM94 are associated with IDDCDF. METHODS AND RESULTS: In a prenatal setting, where fetal abnormalities were detected using antenatal sonography, we used trio-exome sequencing (trio-ES) in conjunction with chromosomal microarray analysis (CMA) to identify two novel homozygous loss of function variants in the TMEM94 gene (c.606dupG and c.2729-2A>G) in two unrelated Saudi Arabian families. CONCLUSIONS: This study provides confirmation that TMEM94 variants may cause IDDCDF. For the first time we describe the pathogenicity of TMEM94 defects detected during the prenatal period.

Renal tubular dysgenesis: antenatal ultrasound scanning and molecular investigations in a Saudi Arabian family.

Autosomal recessive renal tubular dysgenesis (RTD) is a rare lethal disease affecting renal development before birth. RTD is manifested by anuria and severe hypotension resulting in oligohydramnios and birth defects known as Potter's syndrome. Homozygous or compound heterozygous mutations in genes encoding components of the renin-angiotensin system (ACE, AGT, AGTR1 and REN) have been reported to cause RTD. A consanguineous family with a history of multiple stillbirths was investigated using prenatal ultrasound and molecular genetic analysis of an affected foetus. Prenatal ultrasound scan suggested RTD, and a novel homozygous frameshift mutation c.299_300delAA (p.Lys100Serfs*4) in the REN gene was identified by whole-exome sequencing, which segregated with parental DNA samples. RTD remains a rare but important cause of prenatal and perinatal death and may present with antenatally hyperechogenic kidneys.

Identification of embryonic lethal genes in humans by autozygosity mapping and exome sequencing in consanguineous families.

BACKGROUND: Identifying genetic variants that lead to discernible phenotypes is the core of Mendelian genetics. An approach that considers embryonic lethality as a bona fide Mendelian phenotype has the potential to reveal novel genetic causes, which will further our understanding of early human development at a molecular level. Consanguineous families in which embryonic lethality segregates as a recessive Mendelian phenotype offer a unique opportunity for high throughput novel gene discovery as has been established for other recessive postnatal phenotypes. RESULTS: We have studied 24 eligible families using autozygosity mapping and whole-exome sequencing. In addition to revealing mutations in genes previously linked to embryonic lethality in severe cases, our approach revealed seven novel candidate genes (THSD1, PIGC, UBN1, MYOM1, DNAH14, GALNT14, and FZD6). A founder mutation in one of these genes, THSD1, which has been linked to vascular permeability, accounted for embryonic lethality in three of the study families. Unlike the other six candidate genes, we were able to identify a second mutation in THSD1 in a family with a less severe phenotype consisting of hydrops fetalis and persistent postnatal edema, which provides further support for the proposed link between this gene and embryonic lethality. CONCLUSIONS: Our study represents an important step towards the systematic analysis of "embryonic lethal genes" in humans.