Clinical and genetic investigation of 14 families with various forms of short stature syndromes.

Skeletal dysplasias are a heterogeneous group of disorders presenting mild to lethal defects. Several factors, such as genetic, prenatal, and postnatal environmental may contribute to reduced growth. Fourteen families of Pakistani origin, presenting the syndromic form of short stature either in the autosomal recessive or autosomal dominant manner were clinically and genetically investigated to uncover the underlying genetic etiology. Homozygosity mapping, whole exome sequencing, and Sanger sequencing were used to search for the disease-causing gene variants. In total, we have identified 13 sequence variants in 10 different genes. The variants in the HSPG2 and XRCC4 genes were not reported previously in the Pakistani population. This study will expand the mutation spectrum of the identified genes and will help in improved diagnosis of the syndromic form of short stature in the local population.

Loss of symmetric cell division of apical neural progenitors drives DENND5A-related developmental and epileptic encephalopathy.

Developmental and epileptic encephalopathies (DEEs) are a heterogenous group of epilepsies in which altered brain development leads to developmental delay and seizures, with the epileptic activity further negatively impacting neurodevelopment. Identifying the underlying cause of DEEs is essential for progress toward precision therapies. Here we describe a group of individuals with biallelic variants in DENND5A and determine that variant type is correlated with disease severity. We demonstrate that DENND5A interacts with MUPP1 and PALS1, components of the Crumbs apical polarity complex, which is required for both neural progenitor cell identity and the ability of these stem cells to divide symmetrically. Induced pluripotent stem cells lacking DENND5A fail to undergo symmetric cell division during neural induction and have an inherent propensity to differentiate into neurons, and transgenic DENND5A mice, with phenotypes like the human syndrome, have an increased number of neurons in the adult subventricular zone. Disruption of symmetric cell division following loss of DENND5A results from misalignment of the mitotic spindle in apical neural progenitors. A subset of DENND5A is localized to centrosomes, which define the spindle poles during mitosis. Cells lacking DENND5A orient away from the proliferative apical domain surrounding the ventricles, biasing daughter cells towards a more fate-committed state and ultimately shortening the period of neurogenesis. This study provides a mechanism behind DENND5A-related DEE that may be generalizable to other developmental conditions and provides variant-specific clinical information for physicians and families.

ZSCAN10 deficiency causes a neurodevelopmental disorder with characteristic oto-facial malformations.

Neurodevelopmental disorders are major indications for genetic referral and have been linked to more than 1,500 loci including genes encoding transcriptional regulators. The dysfunction of transcription factors often results in characteristic syndromic presentations, however, at least half of these patients lack a genetic diagnosis. The implementation of machine learning approaches has the potential to aid in the identification of new disease genes and delineate associated phenotypes. Next generation sequencing was performed in seven affected individuals with neurodevelopmental delay and dysmorphic features. Clinical characterization included reanalysis of available neuroimaging datasets and 2D portrait image analysis with GestaltMatcher. The functional consequences of ZSCAN10 loss were modelled in mouse embryonic stem cells (mESC), including a knock-out and a representative ZSCAN10 protein truncating variant. These models were characterized by gene expression and Western blot analyses, chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR), and immunofluorescence staining. Zscan10 knockout mouse embryos were generated and phenotyped. We prioritized bi-allelic ZSCAN10 loss-of-function variants in seven affected individuals from five unrelated families as the underlying molecular cause. RNA-Seq analyses in Zscan10-/- mESCs indicated dysregulation of genes related to stem cell pluripotency. In addition, we established in mESCs the loss-of-function mechanism for a representative human ZSCAN10 protein truncating variant by showing alteration of its expression levels and subcellular localization, interfering with its binding to DNA enhancer targets. Deep phenotyping revealed global developmental delay, facial asymmetry, and malformations of the outer ear as consistent clinical features. Cerebral MRI showed dysplasia of the semicircular canals as an anatomical correlate of sensorineural hearing loss. Facial asymmetry was confirmed as a clinical feature by GestaltMatcher and was recapitulated in the Zscan10 mouse model along with inner and outer ear malformations. Our findings provide evidence of a novel syndromic neurodevelopmental disorder caused by bi-allelic loss-of-function variants in ZSCAN10.

Sequence Variants in the WNT10B Underlying Non-Syndromic Split-Hand/Foot Malformation.

Introduction: Split hand and foot malformation (SHFM) or ectrodactyly is a rare limb deformity characterized by median cleft of the hand and foot with impaired or missing central rays. It can occur as an isolated anomaly or in association with abnormalities of other body parts. Methods: After delineating the clinical features of two families (A-B), with non-syndromic SHFM, exome and Sanger sequencing were employed to search for the disease-causing variants. Results: Analysis of exome and Sanger sequencing data revealed two causative variants in the WNT10B gene in affected members of the two families. This included a novel missense change [c.338G>C; p.(Gly113Ala)] in family A and a previously reported frameshift variant [c.884-896delTCCAGCCCCGTCT; p.(Phe295Cysfs*87)] in family B. Conclusion: Our findings add a novel variant in WNT10B gene as the underlying cause of SHFM. The finding adds to the growing body of knowledge about the genetic basis of developmental disorders and provides valuable insights into the molecular mechanisms that regulate limb development.

A PAK1 Mutational Hotspot Within the Regulatory CRIPaK Domain is Associated With Severe Neurodevelopmental Disorders in Children.

BACKGROUND: P-21-activated kinases (PAKs) are protein serine/threonine kinases, part of the RAS/mitogen-activated protein kinase pathway. PAK1 is highly expressed in the central nervous system and crucially involved in neuronal migration and brain developmental processes. Recently, de novo heterozygous missense variants in PAK1 have been identified as an ultrarare cause of pediatric neurodevelopmental disorders. METHODS: We report a series of children affected with postnatal macrocephaly, neurodevelopmental impairment, and drug-resistant epilepsy. Repeated electroencephalographic (EEG) and video-EEG evaluations were performed over a two- to 10-year period during follow-up to delineate electroclinical histories. Genetic sequencing studies and computational evaluation of the identified variants were performed in our patient cohort. RESULTS: We identified by whole-exome sequencing three novel de novo variants in PAK1 (NM_001128620: c.427A>G, p.Met143Val; c.428T>C, p.Met143Thr; c.428T>A, p.Met143Lys) as the underlying cause of the disease in our families. The three variants affected the same highly conserved Met143 residue within the cysteine-rich inhibitor of PAK1 (CRIPaK) domain, which was identified before as a PAK1 inhibitor target. Computational studies suggested a defective autoinhibition presumably due to impaired PAK1 autoregulation as a result of the recurrent substitution. CONCLUSIONS: We delineated the electroclinical phenotypes of PAK1-related neurological disorders and highlight a novel mutational hotspot that may involve defective autoinhibition of the PAK1 protein. The three novel variants affecting the same hotspot residue within the CRIPaK domain highlight potentially impaired PAK1-CRIPaK interaction as a novel disease mechanism. These findings shed light on possible future treatments targeted at the CRIPaK domain, to modulate PAK1 activity and function.

Variants in EFCAB7 underlie nonsyndromic postaxial polydactyly.

Polydactyly is the most common limb malformation that occurs in 1.6-10.6 per one thousand live births, with incidence varying with ancestry. The underlying gene has been identified for many of the ~100 syndromes that include polydactyly. While for the more common form, nonsydromic polydactyly, eleven candidate genes have been reported. We investigated the underlying genetic cause of autosomal recessive nonsyndromic postaxial polydactyly in four consanguineous Pakistani families. Some family members with postaxial polydactyly also present with syndactyly, camptodactyly, or clinodactyly. Analysis of the exome sequence data revealed two novel homozygous frameshift deletions in EFCAB7: [c.830delG;p.(Gly277Valfs*5)]; in three families and [c.1350_1351delGA;p.(Asn451Phefs*2)] in one family. Sanger sequencing confirmed that these variants segregated with postaxial polydactyly, i.e., family members with postaxial polydactyly were found to be homozygous while unaffected members were heterozygous or wild type. EFCAB7 displays expressions in the skeletal muscle and on the cellular level in cilia. IQCE-EFCAB7 and EVC-EVC2 are part of the heterotetramer EvC complex, which is a positive regulator of the Hedgehog (Hh) pathway, that plays a key role in limb formation. Depletion of either EFCAB7 or IQCE inhibits induction of Gli1, a direct Hh target gene. Variants in IQCE and GLI1 have been shown to cause nonsyndromic postaxial polydactyly, while variants in EVC and EVC2 underlie Ellis van Creveld and Weyers syndromes, which include postaxial polydactyly as a phenotype. This is the first report of the involvement of EFCAB7 in human disease etiology.

Clinical trio genome sequencing facilitates the interpretation of variants in cancer predisposition genes in paediatric tumour patients.

The prevalence of pathogenic and likely pathogenic (P/LP) variants in genes associated with cancer predisposition syndromes (CPS) is estimated to be 8-18% for paediatric cancer patients. In more than half of the carriers, the family history is unsuspicious for CPS. Therefore, broad genetic testing could identify germline predisposition in additional children with cancer resulting in important implications for themselves and their families. We thus evaluated clinical trio genome sequencing (TGS) in a cohort of 72 paediatric patients with solid cancers other than retinoblastoma or CNS-tumours. The most prevalent cancer types were sarcoma (n = 26), neuroblastoma (n = 15), and nephroblastoma (n = 10). Overall, P/LP variants in CPS genes were identified in 18.1% of patients (13/72) and P/LP variants in autosomal-dominant CPS genes in 9.7% (7/72). Genetic evaluation would have been recommended for the majority of patients with P/LP variants according to the Jongmans criteria. Four patients (5.6%, 4/72) carried P/LP variants in autosomal-dominant genes known to be associated with their tumour type. With the immediate information on variant inheritance, TGS facilitated the identification of a de novo P/LP in NF1, a gonadosomatic mosaic in WT1 and two pathogenic variants in one patient (DICER1 and PALB2). TGS allows a more detailed characterization of structural variants with base-pair resolution of breakpoints which can be relevant for the interpretation of copy number variants. Altogether, TGS allows comprehensive identification of children with a CPS and supports the individualised clinical management of index patients and high-risk relatives.

Sequence Variants in MEGF8 and GJA1 Underlying Syndactyly.

Introduction: Syndactyly is a common congenital limb malformation. It occurs due to embryological failure of digit separation during limb development. Syndactyly often runs in families with an incidence of about one out of every 2,500-3,000 live births. Methods: Here, we have reported two families presenting features of severe forms of syndactyly. The disorder segregated in autosomal recessive in one and in autosomal dominant manner in the second family. Search for the causative variants was carried out using whole-exome sequencing in family A and candidate gene sequencing in family B. Results: Analysis of the sequencing data revealed two novel missense variants, including p.(Cys1925Arg) in MEGF8 in family A and p.(Thr89Ile) in GJA1 in family B. Conclusion: In conclusion, the novel findings, presented here, not only expand the mutation spectrum in the genes MEGF8 and GJA1, but this will also facilitate screening other families carrying similar clinical features in the Pakistani population.

Genome sequencing identifies complex structural MLH1 variant in unsolved Lynch syndrome.

BACKGROUND: Lynch syndrome is one of the most common cancer predisposition syndromes. It is caused by inherited changes in the mismatch repair pathway. With current diagnostic approaches, a causative genetic variant can be found in less than 50% of cases. A correct diagnosis is important for ensuring that an appropriate surveillance program is used and that additional high-risk family members are identified. METHODS: We used clinical genome sequencing on DNA from blood and subsequent transcriptome sequencing for confirmation. Data were analyzed using the megSAP pipeline and classified according to basic criteria in diagnostic laboratories. Segregation analyses in family members were conducted via breakpoint PCR. RESULTS: We present a family with the clinical diagnosis of Lynch syndrome in which standard diagnostic tests, such as panel or exome sequencing, were unable to detect the underlying genetic variant. Genome sequencing in the index patient confirmed the previous diagnostic results and identified an additional complex rearrangement with intronic breakpoints involving MLH1 and its neighboring gene LRRFIP2. The previously undetected structural variant was classified as medically relevant. Segregation analysis in the family identified additional at-risk individuals which were offered intensified cancer screening. DISCUSSION AND CONCLUSIONS: This case illustrates the advantages of clinical genome sequencing in detecting structural variants compared with current diagnostic approaches. Although structural variants are rare in Lynch syndrome families, they seem to be underreported, in part because of technical challenges. Clinical genome sequencing offers a comprehensive genetic characterization detecting a wide range of genetic variants.

Bi-allelic loss-of-function variants in KIF21A cause severe fetal akinesia with arthrogryposis multiplex.

BACKGROUND: Fetal akinesia (FA) results in variable clinical presentations and has been associated with more than 166 different disease loci. However, the underlying molecular cause remains unclear in many individuals. We aimed to further define the set of genes involved. METHODS: We performed in-depth clinical characterisation and exome sequencing on a cohort of 23 FA index cases sharing arthrogryposis as a common feature. RESULTS: We identified likely pathogenic or pathogenic variants in 12 different established disease genes explaining the disease phenotype in 13 index cases and report 12 novel variants. In the unsolved families, a search for recessive-type variants affecting the same gene was performed; and in five affected fetuses of two unrelated families, a homozygous loss-of-function variant in the kinesin family member 21A gene (KIF21A) was found. CONCLUSION: Our study underlines the broad locus heterogeneity of FA with well-established and atypical genotype-phenotype associations. We describe KIF21A as a new factor implicated in the pathogenesis of severe neurogenic FA sequence with arthrogryposis of multiple joints, pulmonary hypoplasia and facial dysmorphisms. This hypothesis is further corroborated by a recent report on overlapping phenotypes observed in Kif21a null piglets.

Genome Sequencing and Transcriptome Profiling in Twins Discordant for Mayer-Rokitansky-Küster-Hauser Syndrome.

To identify potential genetic causes for Mayer-Rokitansky-Küster-Hauser syndrome (MRKH), we analyzed blood and rudimentary uterine tissue of 5 MRKH discordant monozygotic twin pairs. Assuming that a variant solely identified in the affected twin or affected tissue could cause the phenotype, we identified a mosaic variant in ACTR3B with high allele frequency in the affected tissue, low allele frequency in the blood of the affected twin, and almost absent in blood of the unaffected twin. Focusing on MRKH candidate genes, we detected a pathogenic variant in GREB1L in one twin pair and their unaffected mother showing a reduced phenotypic penetrance. Furthermore, two variants of unknown clinical significance in PAX8 and WNT9B were identified. In addition, we conducted transcriptome analysis of affected tissue and observed perturbations largely similar to those in sporadic cases. These shared transcriptional changes were enriched for terms associated with estrogen and its receptors pointing at a role of estrogen in MRKH pathology. Our genome sequencing approach of blood and uterine tissue of discordant twins is the most extensive study performed on twins discordant for MRKH so far. As no clear pathogenic differences were detected, research to evaluate other regulatory layers are required to better understand the complex etiology of MRKH.

Heterozygous frameshift variants in HNRNPA2B1 cause early-onset oculopharyngeal muscular dystrophy.

Missense variants in RNA-binding proteins (RBPs) underlie a spectrum of disease phenotypes, including amyotrophic lateral sclerosis, frontotemporal dementia, and inclusion body myopathy. Here, we present ten independent families with a severe, progressive muscular dystrophy, reminiscent of oculopharyngeal muscular dystrophy (OPMD) but of much earlier onset, caused by heterozygous frameshift variants in the RBP hnRNPA2/B1. All disease-causing frameshift mutations abolish the native stop codon and extend the reading frame, creating novel transcripts that escape nonsense-mediated decay and are translated to produce hnRNPA2/B1 protein with the same neomorphic C-terminal sequence. In contrast to previously reported disease-causing missense variants in HNRNPA2B1, these frameshift variants do not increase the propensity of hnRNPA2 protein to fibrillize. Rather, the frameshift variants have reduced affinity for the nuclear import receptor karyopherin ß2, resulting in cytoplasmic accumulation of hnRNPA2 protein in cells and in animal models that recapitulate the human pathology. Thus, we expand the phenotypes associated with HNRNPA2B1 to include an early-onset form of OPMD caused by frameshift variants that alter its nucleocytoplasmic transport dynamics.

Human COQ4 deficiency: delineating the clinical, metabolic and neuroimaging phenotypes.

BACKGROUND: Human coenzyme Q4 (COQ4) is essential for coenzyme Q10 (CoQ10) biosynthesis. Pathogenic variants in COQ4 cause childhood-onset neurodegeneration. We aimed to delineate the clinical spectrum and the cellular consequences of COQ4 deficiency. METHODS: Clinical course and neuroradiological findings in a large cohort of paediatric patients with COQ4 deficiency were analysed. Functional studies in patient-derived cell lines were performed. RESULTS: We characterised 44 individuals from 36 families with COQ4 deficiency (16 newly described). A total of 23 different variants were identified, including four novel variants in COQ4. Correlation analyses of clinical and neuroimaging findings revealed three disease patterns: type 1: early-onset phenotype with neonatal brain anomalies and epileptic encephalopathy; type 2: intermediate phenotype with distinct stroke-like lesions; and type 3: moderate phenotype with non-specific brain pathology and a stable disease course. The functional relevance of COQ4 variants was supported by in vitro studies using patient-derived fibroblast lines. Experiments revealed significantly decreased COQ4 protein levels, reduced levels of cellular CoQ10 and elevated levels of the metabolic intermediate 6-demethoxyubiquinone. CONCLUSION: Our study describes the heterogeneous clinical presentation of COQ4 deficiency and identifies phenotypic subtypes. Cell-based studies support the pathogenic characteristics of COQ4 variants. Due to the insufficient clinical response to oral CoQ10 supplementation, alternative treatment strategies are warranted.

Expanding the phenotypic spectrum of FINCA (fibrosis, neurodegeneration, and cerebral angiomatosis) syndrome beyond infancy.

Fibrosis, neurodegeneration, and cerebral angiomatosis (FINCA, MIM#618278) is a rare clinical condition caused by bi-allelic variants in NHL repeat containing protein 2 (NHLRC2, MIM*618277). Pulmonary disease may be the presenting sign and the few patients reported so far, all deceased in early infancy. Exome sequencing was performed on patients with childhood interstitial lung disease (chILD) and additional neurological features. The chILD-EU register database and an in-house database were searched for patients with NHLRC2 variants and clinical features overlapping FINCA syndrome. Six patients from three families were identified with bi-allelic variants in NHLRC2. Two of these children died before the age of two while four others survived until childhood. Interstitial lung disease was pronounced in almost all patients during infancy and stabilized over the course of the disease with neurodevelopmental delay (NDD) evolving as the key clinical finding. We expand the phenotype of FINCA syndrome to a multisystem disorder with variable severity. FINCA syndrome should also be considered in patients beyond infancy with NDD and a history of distinct interstitial lung disease. Managing patients in registers for rare diseases helps identifying new diagnostic entities and advancing care for these patients.

Pitfalls in Genetic Diagnostics: Why Phenotyping is Essential.

New genetic testing technologies have revolutionized medicine within the past years. It is foreseeable that the development will continue with the introduction of new techniques. Nevertheless, despite improved technology, an exact clinical description of the phenotype is still necessary and it is important to critically question findings, both before initiating genetic testing and when interpreting the results. We present four brief case vignettes to point out difficulties associated with correctly interpreting genetic findings.

Bi-allelic HPDL Variants Cause a Neurodegenerative Disease Ranging from Neonatal Encephalopathy to Adolescent-Onset Spastic Paraplegia.

We report bi-allelic pathogenic HPDL variants as a cause of a progressive, pediatric-onset spastic movement disorder with variable clinical presentation. The single-exon gene HPDL encodes a protein of unknown function with sequence similarity to 4-hydroxyphenylpyruvate dioxygenase. Exome sequencing studies in 13 families revealed bi-allelic HPDL variants in each of the 17 individuals affected with this clinically heterogeneous autosomal-recessive neurological disorder. HPDL levels were significantly reduced in fibroblast cell lines derived from more severely affected individuals, indicating the identified HPDL variants resulted in the loss of HPDL protein. Clinical presentation ranged from severe, neonatal-onset neurodevelopmental delay with neuroimaging findings resembling mitochondrial encephalopathy to milder manifestation of adolescent-onset, isolated hereditary spastic paraplegia. All affected individuals developed spasticity predominantly of the lower limbs over the course of the disease. We demonstrated through bioinformatic and cellular studies that HPDL has a mitochondrial localization signal and consequently localizes to mitochondria suggesting a putative role in mitochondrial metabolism. Taken together, these genetic, bioinformatic, and functional studies demonstrate HPDL is a mitochondrial protein, the loss of which causes a clinically variable form of pediatric-onset spastic movement disorder.

Pontocerebellar hypoplasia type 11: Does the genetic defect determine timing of cerebellar pathology?

Pontocerebellar hypoplasia (PCH) comprises a clinically and genetically heterogeneous group of disorders characterized by hypoplasia and degeneration of the cerebellum and ventral pons. To date at least 18 different clinical subtypes of PCH associated with pathogenic variants in 19 different genes have been described. Only recently, bi-allelic variants in TBC1D23 have been reported as the underlying molecular defect in seven index cases with a suspected non-degenerative form of PCH, PCH type 11 (PCH11). We used exome sequencing to investigate an individual with global developmental delay, ataxia, seizures, and progressive PCH. Brain volume was evaluated over a disease course of 14 years using volumetric magnetic resonance imaging (MRI). Volume alterations were compared to age-matched controls as well as data from children with PCH2. We identified a homozygous frameshift variant in exon 9 of 18 of TBC1D23 predicting a loss of protein function. Brain morphometry revealed a pattern of pontine, brain stem, and supratentorial volume loss similar to PCH2 patients although less pronounced. Intriguingly, cerebral MRI findings at the age of 1 and 15 years clearly showed progressive atrophy of the cerebellum, especially the hemispheres. In four of the cases reported in the literature cerebellar hemispheres could be evaluated on the MRIs displayed, they also showed atrophic foliae. While pontine hypoplasia and pronounced microcephaly are in line with previous reports on PCH11, our observations of clearly postnatal atrophy of the cerebellum argues for a different pathomechanism than in the other forms of PCH and supports the hypothesis that TBC1D23 deficiency predominantly interferes with postnatal rather than with prenatal cerebellar development.

First-line exome sequencing in Palestinian and Israeli Arabs with neurological disorders is efficient and facilitates disease gene discovery.

A high rate of consanguinity leads to a high prevalence of autosomal recessive disorders in inbred populations. One example of inbred populations is the Arab communities in Israel and the Palestinian Authority. In the Palestinian Authority in particular, due to limited access to specialized medical care, most patients do not receive a genetic diagnosis and can therefore neither receive genetic counseling nor possibly specific treatment. We used whole-exome sequencing as a first-line diagnostic tool in 83 Palestinian and Israeli Arab families with suspected neurogenetic disorders and were able to establish a probable genetic diagnosis in 51% of the families (42 families). Pathogenic, likely pathogenic or highly suggestive candidate variants were found in the following genes extending and refining the mutational and phenotypic spectrum of these rare disorders: ACO2, ADAT3, ALS2, AMPD2, APTX, B4GALNT1, CAPN1, CLCN1, CNTNAP1, DNAJC6, GAMT, GPT2, KCNQ2, KIF11, LCA5, MCOLN1, MECP2, MFN2, MTMR2, NT5C2, NTRK1, PEX1, POLR3A, PRICKLE1, PRKN, PRX, SCAPER, SEPSECS, SGCG, SLC25A15, SPG11, SYNJ1, TMCO1, and TSEN54. Further, this cohort has proven to be ideal for prioritization of new disease genes. Two separately published candidate genes (WWOX and PAX7) were identified in this study. Analyzing the runs of homozygosity (ROHs) derived from the Exome sequencing data as a marker for the rate of inbreeding, revealed significantly longer ROHs in the included families compared with a German control cohort. The total length of ROHs correlated with the detection rate of recessive disease-causing variants. Identification of the disease-causing gene led to new therapeutic options in four families.