Human ABL1 deficiency syndrome (HADS) is a recognizable syndrome distinct from ABL1-related congenital heart defects and skeletal malformations syndrome.

Germline gain of function variants in the oncogene ABL1 cause congenital heart defects and skeletal malformations (CHDSKM) syndrome. Whether a corresponding ABL1 deficiency disorder exists in humans remains unknown although developmental defects in mice deficient for Abl1 support this notion. Here, we describe two multiplex consanguineous families, each segregating a different homozygous likely loss of function variant in ABL1. The associated phenotype is multiple congenital malformations and distinctive facial dysmorphism that are opposite in many ways to CHDSKM. We suggest that a tight balance of ABL1 activity is required during embryonic development and that both germline gain of function and loss of function variants result in distinctively different allelic congenital malformation disorders.

Genomic analysis of presumed perinatal stroke in Saudi Arabia reveals a strong monogenic contribution.

Perinatal stroke is associated with significant short- and long-term morbidity and has been recognized as the most common cause of cerebral palsy in term infants. The diagnosis of presumed perinatal stroke (PPS) is made in children who present with neurological deficit and/or seizures attributable to focal chronic infarction on neuroimaging and have uneventful neonatal history. The underlying mechanism of presumed perinatal stroke remains unknown and thorough investigation of potential monogenic causes has not been conducted to date. Here, we describe the use of untargeted exome sequencing to investigate a cohort of eight patients from six families with PPS. A likely deleterious variant was identified in four families. These include the well-established risk genes COL4A2 and JAM3. In addition, we report the first independent confirmation of the recently described link between ESAM and perinatal stroke. Our data also highlight NID1 as a candidate gene for the condition. This study suggests that monogenic disorders are important contributors to the pathogenesis of PPS and should be investigated by untargeted sequencing especially when traditional risk factors are excluded.

Large-scale genomic investigation of pediatric cholestasis reveals a novel hepatorenal ciliopathy caused by PSKH1 mutations.

PURPOSE: Pediatric cholestasis is the phenotypic expression of clinically and genetically heterogeneous disorders of bile acid synthesis and flow. Although a growing number of monogenic causes of pediatric cholestasis have been identified, the majority of cases remain undiagnosed molecularly. METHODS: In a cohort of 299 pediatric participants (279 families) with intrahepatic cholestasis, we performed exome sequencing as a first-tier diagnostic test. RESULTS: A likely causal variant was identified in 135 families (48.56%). These comprise 135 families that harbor variants spanning 37 genes with established or tentative links to cholestasis. In addition, we propose a novel candidate gene (PSKH1) (HGNC:9529) in 4 families. PSKH1 was particularly compelling because of strong linkage in 3 consanguineous families who shared a novel hepatorenal ciliopathy phenotype. Two of the 4 families shared a founder homozygous variant, whereas the third and fourth had different homozygous variants in PSKH1. PSKH1 encodes a putative protein serine kinase of unknown function. Patient fibroblasts displayed abnormal cilia that are long and show abnormal transport. A homozygous Pskh1 mutant mouse faithfully recapitulated the human phenotype and displayed abnormally long cilia. The phenotype could be rationalized by the loss of catalytic activity observed for each recombinant PSKH1 variant using in vitro kinase assays. CONCLUSION: Our results support the use of genomics in the workup of pediatric cholestasis and reveal PSKH1-related hepatorenal ciliopathy as a novel candidate monogenic form.

Further delineation of the phenotypic and metabolomic profile of ALDH1L2-related neurodevelopmental disorder.

ALDH1L2, a mitochondrial enzyme in folate metabolism, converts 10-formyl-THF (10-formyltetrahydrofolate) to THF (tetrahydrofolate) and CO2. At the cellular level, deficiency of this NADP+-dependent reaction results in marked reduction in NADPH/NADP+ ratio and reduced mitochondrial ATP. Thus far, a single patient with biallelic ALDH1L2 variants and the phenotype of a neurodevelopmental disorder has been reported. Here, we describe another patient with a neurodevelopmental disorder associated with a novel homozygous missense variant in ALDH1L2, Pro133His. The variant caused marked reduction in the ALDH1L2 enzyme activity in skin fibroblasts derived from the patient as probed by 10-FDDF, a stable synthetic analog of 10-formyl-THF. Additional associated abnormalities in these fibroblasts include reduced NADPH/NADP+ ratio and pool of mitochondrial ATP, upregulated autophagy and dramatically altered metabolomic profile. Overall, our study further supports a link between ALDH1L2 deficiency and abnormal neurodevelopment in humans.

A founder variant expands the phenotype of WNT7B-related PDAC syndrome.

Pulmonary hypoplasia, Diaphragmatic anomalies, Anophthalmia/microphthalmia, and Cardiac defects (PDAC) syndrome is a genetically heterogeneous multiple congenital malformation syndrome. Although pathogenic variants in RARB and STRA6 are established causes of PDAC, many PDAC cases remain unsolved at the molecular level. Recently, we proposed biallelic WNT7B variants as a novel etiology based on several families with typical features of PDAC syndrome albeit with variable expressivity. Here, we report three patients from two families that share a novel founder variant in WNT7B (c.739C > T; Arg247Trp). The phenotypic expression of this variant ranges from typical PDAC features to isolated genitourinary anomalies. Similar to previously reported PDAC-associated WNT7B variants, this variant was found to significantly impair WNT7B signaling activity further corroborating its proposed pathogenicity. This report adds further evidence to WNT7B-related PDAC and expands its variable expressivity.

Homozygous truncating variant in MAN2A2 causes a novel congenital disorder of glycosylation with neurological involvement.

BACKGROUND: Enzymes of the Golgi implicated in N-glycan processing are critical for brain development, and defects in many are defined as congenital disorders of glycosylation (CDG). Involvement of the Golgi mannosidase, MAN2A2 has not been identified previously as causing glycosylation defects. METHODS: Exome sequencing of affected individuals was performed with Sanger sequencing of the MAN2A2 transcript to confirm the variant. N-glycans were analysed in patient-derived lymphoblasts to determine the functional effects of the variant. A cell-based complementation assay was designed to assess the pathogenicity of identified variants using MAN2A1/MAN2A2 double knock out HEK293 cell lines. RESULTS: We identified a multiplex consanguineous family with a homozygous truncating variant p.Val1101Ter in MAN2A2. Lymphoblasts from two affected brothers carrying the same truncating variant showed decreases in complex N-glycans and accumulation of hybrid N-glycans. On testing of this variant in the developed complementation assay, we see the complete lack of complex N-glycans. CONCLUSION: Our findings show that pathogenic variants in MAN2A2 cause a novel autosomal recessive CDG with neurological involvement and facial dysmorphism. Here, we also present the development of a cell-based complementation assay to assess the pathogenicity of MAN2A2 variants, which can also be extended to MAN2A1 variants for future diagnosis.

PRSS8, encoding prostasin, is mutated in patients with autosomal recessive ichthyosis.

Ichthyosis is a genetically heterogeneous genodermatosis characterized by severely rough, dry and scaly skin. We report two consanguineous families with congenital ichthyosis. Combined positional mapping and exome sequencing of the two families revealed novel homozygous likely deleterious variants in PRSS8 (encoding prostasin) within a linkage locus on chromosome 16. One variant involved a canonical splice site and was associated with reduced abundance of the normal transcript, while the other was a missense variant that altered a highly conserved residue. The phenotype of Prss8 knockout mouse bears a striking resemblance to the one we describe in human patients, including the skin histopathology. Our data suggest a novel PRSS8-related ichthyosis disorder.

A founder DBR1 variant causes a lethal form of congenital ichthyosis.

DBR1 encodes the only known human lariat debranching enzyme and its deficiency has been found to cause an autosomal recessive inborn error of immunity characterized by pediatric brainstem viral-induced encephalitis (MIM 619441). We describe a distinct allelic disorder caused by a founder recessive DBR1 variant in four families (DBR1(NM_016216.4):c.200A > G (p.Tyr67Cys)). Consistent features include prematurity, severe intrauterine growth deficiency, congenital ichthyosis-like presentation (collodion membrane, severe skin peeling and xerosis), and death before the first year of life. Patient-derived fibroblasts displayed the characteristic accumulation of intron lariats in their RNA as revealed by targeted and untargeted analysis, in addition to a marked reduction of DBR1 on immunoblot analysis. We propose a novel DBR1-related developmental disorder that is distinct from DBR1-related encephalitis susceptibility and highlight the apparent lack of correlation with the degree of DBR1 deficiency.

Human 'knockouts' of CSF3 display severe congenital neutropenia.

Colony-stimulating factor 3 (CSF3) is a key factor in neutrophil production and function, and recombinant forms have been used clinically for decades to treat congenital and acquired neutropenia. Although biallelic inactivation of its receptor CSF3R is a well-established cause of severe congenital neutropenia (SCN), no corresponding Mendelian disease has been ascribed to date to CSF3. Here, we describe three patients from two families each segregating a different biallelic inactivating variant in CSF3 with SCN. Complete deficiency of CSF3 as a result of nonsense-mediated decay (NMD) could be demonstrated on RT-PCR using skin fibroblasts-derived RNA. The phenotype observed in this cohort mirrors that documented in mouse and zebrafish models of CSF3 deficiency. Our results suggest that CSF3 deficiency in humans causes a novel autosomal recessive form of SCN.

Mitochondrial "dysmorphology" in variant classification.

Mitochondrial disorders are challenging to diagnose. Exome sequencing has greatly enhanced the diagnostic precision of these disorders although interpreting variants of uncertain significance (VUS) remains a formidable obstacle. Whether specific mitochondrial morphological changes can aid in the classification of these variants is unknown. Here, we describe two families (four patients), each with a VUS in a gene known to affect the morphology of mitochondria through a specific role in the fission-fusion balance. In the first, the missense variant in MFF, encoding a fission factor, was associated with impaired fission giving rise to a characteristically over-tubular appearance of mitochondria. In the second, the missense variant in DNAJA3, which has no listed OMIM phenotype, was associated with fragmented appearance of mitochondria consistent with its published deficiency states. In both instances, the highly specific phenotypes allowed us to upgrade the classification of the variants. Our results suggest that, in select cases, mitochondrial "dysmorphology" can be helpful in interpreting variants to reach a molecular diagnosis.

Bi-allelic Mutations in FAM149B1 Cause Abnormal Primary Cilium and a Range of Ciliopathy Phenotypes in Humans.

Ciliopathies are clinical disorders of the primary cilium with widely recognized phenotypic and genetic heterogeneity. In two Arab consanguineous families, we mapped a ciliopathy phenotype that most closely matches Joubert syndrome (hypotonia, developmental delay, typical facies, oculomotor apraxia, polydactyly, and subtle posterior fossa abnormalities) to a single locus in which a founder homozygous truncating variant in FAM149B1 was identified by exome sequencing. We subsequently identified a third Arab consanguineous multiplex family in which the phenotype of Joubert syndrome/oral-facial-digital syndrome (OFD VI) was found to co-segregate with the same founder variant in FAM149B1. Independently, autozygosity mapping and exome sequencing in a consanguineous Turkish family with Joubert syndrome highlighted a different homozygous truncating variant in the same gene. FAM149B1 encodes a protein of unknown function. Mutant fibroblasts were found to have normal ciliogenesis potential. However, distinct cilia-related abnormalities were observed in these cells: abnormal accumulation IFT complex at the distal tips of the cilia, which assumed bulbous appearance, increased length of the primary cilium, and dysregulated SHH signaling. We conclude that FAM149B1 is required for normal ciliary biology and that its deficiency results in a range of ciliopathy phenotypes in humans along the spectrum of Joubert syndrome.

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.

PLXNA2 as a candidate gene in patients with intellectual disability.

Intellectual disability (ID) is one of the most common disabilities in humans. In an effort to contribute to the expanding genetic landscape of ID, we describe a novel autosomal recessive ID candidate gene. Combined autozygome/exome analysis was performed in two unrelated consanguineous families with ID. Each of the two families had a novel homozygous likely deleterious variant in PLXNA2 and displayed the core phenotype of ID. PLXNA2 belongs to a family of transmembrane proteins that function as semaphorin receptors. Sema5A-PlexinA2 is known to regulate brain development in mouse, and Plxna2-/- mice display defective associative learning, sociability, and sensorimotor gating. We note the existence of variability in the phenotype among the three patients, including the existence of variable degree of ID, ranging from borderline intellectual functioning to moderate-severe ID, and the presence of cardiac anomalies in only one of the patients. We propose incomplete penetrance as a possible explanation of the observed difference in phenotypes. Future cases will be needed to support the proposed link between PLXNA2 and ID in humans.

A genomics approach to females with infertility and recurrent pregnancy loss.

Infertility affects 10% of reproductive-age women and is extremely heterogeneous in etiology. The genetic contribution to female infertility is incompletely understood, and involves chromosomal and single-gene defects. Our aim in this study is to decipher single-gene causes in infertile women in whom endocrinological, anatomical, and chromosomal causes have been excluded. Our cohort comprises women with recurrent pregnancy loss and no offspring from spontaneous pregnancies (RPL, n = 61) and those who never achieved clinical pregnancy and were referred for in vitro fertilization [primary infertility (PI), n = 14]. Whole-exome sequencing revealed candidate variants in 14, which represents 43% of those with PI and 13% of those with RPL. These include variants in previously established female infertility-related genes (TLE6, NLRP7, FSHR, and ZP1) as well as genes with only tentative links in the literature (NLRP5). Candidate variants in genes linked to primary ciliary dyskinesia (DNAH11 and CCNO) were identified in individuals with and without systemic features of the disease. We also identified variants in genes not previously linked to female infertility. These include one homozygous variant each in CCDC68, CBX3, CENPH, PABPC1L, PIF1, PLK1, and REXO4, which we propose as candidate genes for infertility based on their established biology or compatible animal models. Our study expands the contribution of single genes to the etiology of PI and RPL, improves the precision of disease classification at the molecular level, and offers the potential for future treatment and development of human genetics-inspired fertility regulators.

An exome-first approach to aid in the diagnosis of primary ciliary dyskinesia.

Unlike disorders of primary cilium, primary ciliary dyskinesia (PCD) has a much narrower clinical spectrum consistent with the limited tissue distribution of motile cilia. Nonetheless, PCD diagnosis can be challenging due to the overlapping features with other disorders and the requirement for sophisticated tests that are only available in specialized centers. We performed exome sequencing on all patients with a clinical suspicion of PCD but for whom no nasal nitric oxide test or ciliary functional assessment could be ordered. Among 81 patients (56 families), in whom PCD was suspected, 68% had pathogenic or likely pathogenic variants in established PCD-related genes that fully explain the phenotype (20 variants in 11 genes). The major clinical presentations were sinopulmonary infections (SPI) (n = 58), neonatal respiratory distress (NRD) (n = 2), laterality defect (LD) (n = 6), and combined LD/SPI (n = 15). Biallelic likely deleterious variants were also encountered in AKNA and GOLGA3, which we propose as novel candidates in a lung phenotype that overlaps clinically with PCD. We also encountered a PCD phenocopy caused by a pathogenic variant in ITCH, and a pathogenic variant in CEP164 causing Bardet-Biedl syndrome and PCD presentation as a very rare example of the dual presentation of these two disorders of the primary and motile cilia. Exome sequencing is a powerful tool that can help "democratize" the diagnosis of PCD, which is currently limited to highly specialized centers.

Female Infertility Caused by Mutations in the Oocyte-Specific Translational Repressor PATL2.

Infertility is a relatively common disorder of the reproductive system and remains unexplained in many cases. In vitro fertilization techniques have uncovered previously unrecognized infertility phenotypes, including oocyte maturation arrest, the molecular etiology of which remains largely unknown. We report two families affected by female-limited infertility caused by oocyte maturation failure. Positional mapping and whole-exome sequencing revealed two homozygous, likely deleterious variants in PATL2, each of which fully segregates with the phenotype within the respective family. PATL2 encodes a highly conserved oocyte-specific mRNP repressor of translation. Previous data have shown the strict requirement for PATL2 in oocyte-maturation in model organisms. Data gathered from the families in this study suggest that the role of PATL2 is conserved in humans and expand our knowledge of the factors that are necessary for female meiosis.

GZF1 Mutations Expand the Genetic Heterogeneity of Larsen Syndrome.

Larsen syndrome is characterized by the dislocation of large joints and other less consistent clinical findings. Heterozygous FLNB mutations account for the majority of Larsen syndrome cases, but biallelic mutations in CHST3 and B4GALT7 have been more recently described, thus confirming the existence of recessive forms of the disease. In a multiplex consanguineous Saudi family affected by severe and recurrent large joint dislocation and severe myopia, we identified a homozygous truncating variant in GZF1 through a combined autozygome and exome approach. Independently, the same approach identified a second homozygous truncating GZF1 variant in another multiplex consanguineous family affected by severe myopia, retinal detachment, and milder skeletal involvement. GZF1 encodes GDNF-inducible zinc finger protein 1, a transcription factor of unknown developmental function, which we found to be expressed in the eyes and limbs of developing mice. Global transcriptional profiling of cells from affected individuals revealed a shared pattern of gene dysregulation and significant enrichment of genes encoding matrix proteins, including P3H2, which hints at a potential disease mechanism. Our results suggest that GZF1 mutations cause a phenotype of severe myopia and significant articular involvement not previously described in Larsen syndrome.

A genomics approach to male infertility.

PURPOSE: Male infertility remains poorly understood at the molecular level. We aimed in this study to investigate the yield of a "genomics first" approach to male infertility. METHODS: Patients with severe oligospermia and nonobstructive azoospermia were investigated using exome sequencing (ES) in parallel with the standard practice of chromosomal analysis. RESULTS: In 285 patients, 10.5% (n = 30) had evidence of chromosomal aberrations while nearly a quarter (n = 69; 24.2%) had a potential monogenic form of male infertility. The latter ranged from variants in genes previously reported to cause male infertility with or without other phenotypes in humans (24 patients; 8.4%) to those in novel candidate genes reported in this study (37 patients; 12.9%). The 33 candidate genes have biological links to male germ cell development including compatible mouse knockouts, and a few (TERB1 [CCDC79], PIWIL2, MAGEE2, and ZSWIM7) were found to be independently mutated in unrelated patients in our cohort. We also found that male infertility can be the sole or major phenotypic expression of a number of genes that are known to cause multisystemic manifestations in humans (n = 9 patients; 3.1%). CONCLUSION: The standard approach to male infertility overlooks the significant contribution of monogenic causes to this important clinical entity.

The morbid genome of ciliopathies: an update.

PURPOSE: Ciliopathies are highly heterogeneous clinical disorders of the primary cilium. We aim to characterize a large cohort of ciliopathies phenotypically and molecularly. METHODS: Detailed phenotypic and genomic analysis of patients with ciliopathies, and functional characterization of novel candidate genes. RESULTS: In this study, we describe 125 families with ciliopathies and show that deleterious variants in previously reported genes, including cryptic splicing variants, account for 87% of cases. Additionally, we further support a number of previously reported candidate genes (BBIP1, MAPKBP1, PDE6D, and WDPCP), and propose nine novel candidate genes (CCDC67, CCDC96, CCDC172, CEP295, FAM166B, LRRC34, TMEM17, TTC6, and TTC23), three of which (LRRC34, TTC6, and TTC23) are supported by functional assays that we performed on available patient-derived fibroblasts. From a phenotypic perspective, we expand the phenomenon of allelism that characterizes ciliopathies by describing novel associations including WDR19-related Stargardt disease and SCLT1- and CEP164-related Bardet-Biedl syndrome. CONCLUSION: In this cohort of phenotypically and molecularly characterized ciliopathies, we draw important lessons that inform the clinical management and the diagnostics of this class of disorders as well as their basic biology.

Confirming the recessive inheritance of PERP-related erythrokeratoderma.

Erythrokeratoderma (EK) is heterogeneous clinical entity characterized by excessive scaling with resulting erythrokeratotic plaques. Several genes have been linked to EK and they encode a number of proteins that are important for the integrity of the keratinocyte layer of the epidermis. PERP is a transcription factor that is activated by both p53 and p63. However, its deficiency in a mouse model appears to only recapitulate p63-mediated role in skin development and organization. We report an extended multiplex consanguineous family in which an EK phenotype with a striking similarity to that observed in Perp-/- mice, is mapped to an autozygous region on chromosome 6 that spans PERP. Whole-exome sequencing revealed a novel variant in PERP that fully segregated with the phenotype. Functional analysis of patient- and control-derived keratinocytes revealed a deleterious effect of the identified variant on the intracellular localization of PERP. A previous report showed that PERP mutation causes a dominant form of keratoderma but a single patient in that report with a homozygous variant in PERP suggests that recessive inheritance is also possible. Our results, therefore, support the establishment of an autosomal recessive PERP-related EK phenotype in humans.