PRIM1 deficiency causes a distinctive primordial dwarfism syndrome.

DNA replication is fundamental for cell proliferation in all organisms. Nonetheless, components of the replisome have been implicated in human disease, and here we report PRIM1 encoding the catalytic subunit of DNA primase as a novel disease gene. Using a variant classification agnostic approach, biallelic mutations in PRIM1 were identified in five individuals. PRIM1 protein levels were markedly reduced in patient cells, accompanied by replication fork asymmetry, increased interorigin distances, replication stress, and prolonged S-phase duration. Consequently, cell proliferation was markedly impaired, explaining the patients' extreme growth failure. Notably, phenotypic features distinct from those previously reported with DNA polymerase genes were evident, highlighting differing developmental requirements for this core replisome component that warrant future investigation.

Mutation of the Thap4 gene causes dwarfism and testicular anomalies in rats and mice.

The petit (pet) locus is associated with dwarfism, testicular anomalies, severe thymic hypoplasia, and high postnatal lethality, which are inherited in autosomal recessive mode of inheritance in rats with a Wistar strain genetic background. Linkage analysis localized the pet locus between 98.7 Mb and 101.2 Mb on rat chromosome 9. Nucleotide sequence analysis identified 2 bp deletion in exon 2 of the Thap4 gene as the causative mutation for pet. This deletion causes a frameshift and premature termination codon, resulting in a truncated THAP4 protein lacking approximately two-thirds of the C-terminal side. Thap4 is expressed in various organs, including the testis and thymus in rats. To elucidate the biological function of THAP4 in other species, we generated Thap4 knockout mice lacking exon 2 of the Thap4 gene through genome editing. Thap4 knockout mice also exhibited dwarfism and small testis but did not show high postnatal lethality. Thymus weights of adult Thap4 knockout male mice were significantly higher compared to wild-type male mice. Although Thap4 knockout male mice were fertile, their testis contained seminiferous tubules with spermatogenesis and degenerative seminiferous tubules lacking germ cells. Additionally, we observed vacuoles in seminiferous tubules, and clusters of cells in the lumen in seminiferous tubules in Thap4 knockout male mice. These results demonstrate that spontaneous mutation of Thap4 gene in rats and knockout of Thap4 gene in mice both cause dwarfism and testicular anomalies. Thap4 gene in rats and mice is essential for normal testicular development, maintaining spermatogenesis throughout the entire region of seminiferous tubules.

Novel biallelic PISD missense variants cause spondyloepimetaphyseal dysplasia with disproportionate short stature and fragmented mitochondrial morphology.

Biallelic variants in PISD cause a phenotypic spectrum ranging from short stature with spondyloepimetaphyseal dysplasia (SEMD) to a multisystem disorder affecting eyes, ears, bones, and brain. PISD encodes the mitochondrial-localized enzyme phosphatidylserine decarboxylase. The PISD precursor is self-cleaved to generate a heteromeric mature enzyme that converts phosphatidylserine to the phospholipid phosphatidylethanolamine. We describe a 17-year-old male patient, born to unrelated healthy parents, with disproportionate short stature and SEMD, featuring platyspondyly, prominent epiphyses, and metaphyseal dysplasia. Trio genome sequencing revealed compound heterozygous PISD variants c.569C>T; p.(Ser190Leu) and c.799C>T; p.(His267Tyr) in the patient. Investigation of fibroblasts showed similar levels of the PISD precursor protein in both patient and control cells. However, patient cells had a significantly higher proportion of fragmented mitochondria compared to control cells cultured under basal condition and after treatment with 2-deoxyglucose that represses glycolysis and stimulates respiration. Structural data from the PISD orthologue in Escherichia coli suggest that the amino acid substitutions Ser190Leu and His267Tyr likely impair PISD's autoprocessing activity and/or phosphatidylethanolamine biosynthesis. Based on the data, we propose that the novel PISD p.(Ser190Leu) and p.(His267Tyr) variants likely act as hypomorphs and underlie the pure skeletal phenotype in the patient.

RMRP-related short stature: A report of six additional Japanese individuals with cartilage hair hypoplasia and literature review.

Biallelic pathogenic variants in RMRP, the gene encoding the RNA component of RNase mitochondrial RNA processing enzyme complex, have been reported in individuals with cartilage hair hypoplasia (CHH). CHH is prevalent in Finnish and Amish populations due to a founder pathogenic variant, n.71A > G. Based on the manifestations in the Finnish and Amish individuals, the hallmarks of CHH are prenatal-onset growth failure, metaphyseal dysplasia, hair hypoplasia, immunodeficiency, and other extraskeletal manifestations. Herein, we report six Japanese individuals with CHH from four families. All probands presented with moderate short stature with mild metaphyseal dysplasia or brachydactyly. One of them had hair hypoplasia and the other immunodeficiency. By contrast, the affected siblings of two families showed only mild short stature. We also reviewed all previously reported 13 Japanese individuals. No n.71A > G allele was detected. The proportions of Japanese versus Finnish individuals were 0% versus 70% for birth length < -2.0 SD, 84% versus 100% for metaphyseal dysplasia and 26% versus 88% for hair hypoplasia. Milder manifestations in the Japanese individuals may be related to the difference of genotypes. The mildest form of CHH phenotypes is mild short stature without overt skeletal alteration or extraskeletal manifestation and can be termed "RMRP-related short stature".

De Novo Variants in LMNB1 Cause Pronounced Syndromic Microcephaly and Disruption of Nuclear Envelope Integrity.

Lamin B1 plays an important role in the nuclear envelope stability, the regulation of gene expression, and neural development. Duplication of LMNB1, or missense mutations increasing LMNB1 expression, are associated with autosomal-dominant leukodystrophy. On the basis of its role in neurogenesis, it has been postulated that LMNB1 variants could cause microcephaly. Here, we confirm this hypothesis with the identification of de novo mutations in LMNB1 in seven individuals with pronounced primary microcephaly (ranging from -3.6 to -12 SD) associated with relative short stature and variable degree of intellectual disability and neurological features as the core symptoms. Simplified gyral pattern of the cortex and abnormal corpus callosum were noted on MRI of three individuals, and these individuals also presented with a more severe phenotype. Functional analysis of the three missense mutations showed impaired formation of the LMNB1 nuclear lamina. The two variants located within the head group of LMNB1 result in a decrease in the nuclear localization of the protein and an increase in misshapen nuclei. We further demonstrate that another mutation, located in the coil region, leads to increased frequency of condensed nuclei and lower steady-state levels of lamin B1 in proband lymphoblasts. Our findings collectively indicate that de novo mutations in LMNB1 result in a dominant and damaging effect on nuclear envelope formation that correlates with microcephaly in humans. This adds LMNB1 to the growing list of genes implicated in severe autosomal-dominant microcephaly and broadens the phenotypic spectrum of the laminopathies.

A budding yeast model for human disease mutations in the EXOSC2 cap subunit of the RNA exosome complex.

RNA exosomopathies, a growing family of diseases, are linked to missense mutations in genes encoding structural subunits of the evolutionarily conserved, 10-subunit exoribonuclease complex, the RNA exosome. This complex consists of a three-subunit cap, a six-subunit, barrel-shaped core, and a catalytic base subunit. While a number of mutations in RNA exosome genes cause pontocerebellar hypoplasia, mutations in the cap subunit gene EXOSC2 cause an apparently distinct clinical presentation that has been defined as a novel syndrome SHRF (short stature, hearing loss, retinitis pigmentosa, and distinctive facies). We generated the first in vivo model of the SHRF pathogenic amino acid substitutions using budding yeast by modeling pathogenic EXOSC2 missense mutations (p.Gly30Val and p.Gly198Asp) in the orthologous S. cerevisiae gene RRP4 The resulting rrp4 mutant cells show defects in cell growth and RNA exosome function. Consistent with altered RNA exosome function, we detect significant transcriptomic changes in both coding and noncoding RNAs in rrp4-G226D cells that model EXOSC2 p.Gly198Asp, suggesting defects in nuclear surveillance. Biochemical and genetic analyses suggest that the Rrp4 G226D variant subunit shows impaired interactions with key RNA exosome cofactors that modulate the function of the complex. These results provide the first in vivo evidence that pathogenic missense mutations present in EXOSC2 impair the function of the RNA exosome. This study also sets the stage to compare exosomopathy models to understand how defects in RNA exosome function underlie distinct pathologies.

Biallelic variants in the RNA exosome gene EXOSC5 are associated with developmental delays, short stature, cerebellar hypoplasia and motor weakness.

The RNA exosome is an essential ribonuclease complex required for processing and/or degradation of both coding and non-coding RNAs. We identified five patients with biallelic variants in EXOSC5, which encodes a structural subunit of the RNA exosome. The clinical features of these patients include failure to thrive, short stature, feeding difficulties, developmental delays that affect motor skills, hypotonia and esotropia. Brain MRI revealed cerebellar hypoplasia and ventriculomegaly. While we ascertained five patients, three patients with distinct variants of EXOSC5 were studied in detail. The first patient had a deletion involving exons 5-6 of EXOSC5 and a missense variant, p.Thr114Ile, that were inherited in trans, the second patient was homozygous for p.Leu206His and the third patient had paternal isodisomy for chromosome 19 and was homozygous for p.Met148Thr. The additional two patients ascertained are siblings who had an early frameshift mutation in EXOSC5 and the p.Thr114Ile missense variant that were inherited in trans. We employed three complementary approaches to explore the requirement for EXOSC5 in brain development and assess consequences of pathogenic EXOSC5 variants. Loss of function for exosc5 in zebrafish results in shortened and curved tails/bodies, reduced eye/head size and edema. We modeled pathogenic EXOSC5 variants in both budding yeast and mammalian cells. Some of these variants cause defects in RNA exosome function as well as altered interactions with other RNA exosome subunits. These findings expand the number of genes encoding RNA exosome subunits linked to human disease while also suggesting that disease mechanism varies depending on the specific pathogenic variant.

Microcephalic primordial dwarfism with predominant Meier-Gorlin phenotype, ichthyosis, and multiple joint deformities-Further expansion of DONSON Cell Cycle-opathy phenotypic spectrum.

We report a patient with microcephalic primordial dwarfism with predominant Meier-Gorlin syndrome phenotype with ichthyosis and disabling multiple joint deformities in addition to classic features of the syndrome. The patient was a 10.5-year-old girl referred in view of short stature, joint deformities, and facial dysmorphism. There was history of intrauterine growth restriction and collodion like skin abnormality at birth. She had normal developmental milestones and intellect. On clinical evaluation, anthropometry was suggestive of proportionate short stature and microcephaly. There was abnormal posture due to spine and peripheral joint deformities, along with ichthyosis, facial, and digital dysmorphism. Skeletal radiographs showed radial subluxation, acetabular dysplasia and hip dislocation, bilateral knee joint dislocation, absent patellae, slender long bones with delayed bone age, and subluxation of small joints of hands and feet. Work up for metabolic bone disease and peripheral blood karyotype was normal. Whole exome sequencing revealed a pathogenic homozygous variant c.C1297T (p.Pro433Ser) in the exon 8 of DONSON gene. This report further expands the genotypic-phenotypic spectrum of the group of disorders known as Cell Cycle-opathies.

Short stature explained by dimerization of human growth hormone induced by a p.C53S point mutation.

A homozygous mutation in growth hormone 1 (GH1) was recently identified in an individual with growth failure. This mutation, c.705G>C, causes replacement of cysteine at position 53 of the 191-amino-acid sequence of 22 kDa human GH (hGH) with serine (p.C53S). This hGH molecule (hereafter referred to as GH-C53S) lacks the disulfide bond between p.Cys-53 and p.Cys-165, which is highly conserved among species. It has been reported previously that monomeric GH-C53S has reduced bioactivity compared with WT GH (GH-WT) because of its decreased ability to bind and activate the GH receptor in vitro In this study, we discovered that substitution of p.Cys-53 in hGH significantly increased formation of hGH dimers in pituitary cells. We expressed His-tagged hGH variants in the cytoplasm of genetically modified Rosetta-gami B DE3 Escherichia coli cells, facilitating high-yield production. We observed that the bioactivity of monomeric GH-C53S is 25.2% of that of GH-WT and that dimeric GH-C53S-His has no significant bioactivity in cell proliferation assays. We also found that the expression of GH-C53S in pituitary cells deviates from that of GH-WT. GH-C53S was exclusively stained in the Golgi apparatus, and no secretory granules formed for this variant, impairing its stimulated release. In summary, the unpaired Cys-165 in GH-C53S forms a disulfide bond linking two hGH molecules in pituitary cells. We conclude that the GH-C53S dimer is inactive and responsible for the growth failure in the affected individual.

Characterization of a complex phenotype (fever-dependent recurrent acute liver failure and osteogenesis imperfecta) due to NBAS and P4HB variants.

We report the clinical, biochemical and genetic findings from a Spanish boy of Caucasian origin who presented with fever-dependent RALF (recurrent acute liver failure) and osteogenesis imperfecta (OI). Whole-exome sequencing (WES) uncovered two compound heterozygous variants in NBAS (c.[1265 T > C];[1549C > T]:p.[(Leu422Pro)];[(Arg517Cys)]), and a heterozygous variant in P4HB (c.[194A > G];[194=]:p.[(Lys65Arg)];[(Lys65=)]) that was transmitted from the clinically unaffected mother who was mosaic carrier of the variant. Variants in NBAS protein have been associated with ILFS2 (infantile liver failure syndrome-2), SOPH syndrome (short stature, optic nerve atrophy, and Pelger-Huët anomaly syndrome), and multisystem diseases. Several patients showed clinical manifestations affecting the skeletal system, such as osteoporosis, pathologic fractures and OI. Experiments in the patient's fibroblasts demonstrated that mutated NBAS protein is overexpressed and thermally unstable, and reduces the expression of MGP, a regulator of bone homeostasis. Variant in PDI (protein encoded by P4HB) has been associated with CLCRP1 (Cole-Carpenter syndrome-1), a type of severe OI. An increase of COL1A2 protein retention was observed in the patient's fibroblasts. In order to study if the variant in P4HB was involved in the alteration in collagen trafficking, overexpression experiments of PDI were carried out. These experiments showed that overexpression of mutated PDI protein produces an increase in COL1A2 retention. In conclusion, these results corroborate that the variants in NBAS are responsible for the liver phenotype, and demonstrate that the variant in P4HB is involved in the bone phenotype, probably in synergy with NBAS variants.

Age at and indication for diagnosis of Turner syndrome in the pediatric population.

Early diagnosis of Turner syndrome (TS) enables timely intervention and may improve outcomes, but many are still diagnosed late. The objectives of our study were to describe the age and clinical features leading to diagnosis of TS in a large referral center. We hypothesize that newer testing modalities, such as noninvasive prenatal testing (NIPT), may lead to a decline in the age of diagnosis. Medical records of TS patients followed at The Cincinnati Center for Pediatric and Adult TS Care between 1997 and 2016 were reviewed for age at diagnosis, karyotype, and clinical indication(s). Patients (<18 years) were included (n = 239). Thirty-seven percent of patients were diagnosed prenatally or neonatally (≤1 month). The median age of diagnosis was 1.5 (IQR 0.0-10.0) years. If not made during those periods, the median age was 9.3 (IQR 3.2-12.5) years. The most common indications for diagnosis were before birth, unspecified prenatal testing (57%); in neonates/infants, lymphedema (21%); in childhood, short stature (72%); and in adolescence, short stature (45%) followed by pubertal delay with short stature (22%). The age of TS diagnosis in our cohort is young. However, when the diagnosis is not made before 1 year, the median age of diagnosis has not changed in recent years. The age at diagnosis could decrease with prenatal testing, although our study may not have assessed a long enough period of increased use of NIPT. Together with an increase in provider clinical awareness, this may result in more age-appropriate screening of comorbidities and earlier therapeutic intervention.

Alazami syndrome: Report of three Indian patients with phenotypic spectrum from adolescence to adulthood.

Alazami syndrome (ALAZS) (MIM 615071) is a rare autosomal recessive disorder characterized by short stature, dysmorphic facial features, developmental delay, and impaired intellect. It was first reported in a Saudi Arabian family in 2012. Three Indian patients affected with ALAZS, one boy aged 13 years and other two sisters in their 40s are presented. These patients had few unreported dysmorphic facial features: high arched eyebrows and dental overcrowding. No microcephaly was noted in the sisters. One of the sisters did not have short stature. The boy also presented with unilateral buphthalmos of left eye. All three of them have been identified to harbor novel variants in LARP7.

Spondyloepimetaphyseal dysplasia EXTL3-deficient type: Long-term follow-up and review of the literature.

Spondyloepimetaphyseal dysplasia (SEMD) is a group of genetic skeletal disorders characterized by disproportionate short stature, and varying degrees of vertebral, epiphyseal, and metaphyseal involvement of the skeleton. According to the Nosology and classification of genetic skeletal disorders 2019 revision, more than 20 types of SEMD have been identified, and SEMD with immune deficiency, EXTL3 type is one of the newcomers. Affected individuals display variable skeletal abnormalities and neurodevelopmental findings. Liver and kidney cysts have also been reported frequently. Patients may exhibit varying degrees of immune deficiency as well. To date, only 14 patients from 9 unrelated families with SEMD with immune deficiency, EXTL3 type have been reported in the literature. We report a new patient who is currently 15 years old in whom cystic liver lesions were detected in the prenatal period. Disproportionate short stature, mild developmental delay and a T- NK+ B+ immunological profile were detected in the postnatal follow-up. Exome sequence analysis revealed a previously reported homozygous missense variant in exon 3 c.953C > T; p.(Pro318Leu) in EXTL3.

Two loss-of-function ANKRD11 variants in Chinese patients with short stature and a possible molecular pathway.

KBG syndrome is a rare genetic disease characterized mainly by skeletal abnormalities, distinctive facial features, and intellectual disability. Heterozygous mutations in ANKRD11 gene, or deletion of 16q24.3 that includes ANKRD11 gene are the cause of KBG syndrome. We describe two patients presenting with short stature and partial facial features, whereas no intellectual disability or hearing loss was observed in them. Two ANKRD11 variants, c.4039_4041del (p. Lys1347del) and c.6427C > G (p. Leu2143Val), were identified in this study. Both of them were classified as variants of uncertain significance (VOUS) by ACMG/AMP guidelines and were inherited from their mothers. ANKRD11 could enhance the transactivation of p21 gene, which was identified to participate in chondrogenic differentiation. In this study, we demonstrated that the knockdown of ANKRD11 could reduce the p21-promoter luciferase activities while re-introduction of wild type ANKRD11, but not ANKRD11 variants (p. Lys1347del or p. Leu2143Val), could restore the p21 levels. Thus, our study report two loss-of-function ANKRD11 variants which might provide new insight on pathogenic mechanism that correlates ANKRD11 variants with the short stature phenotype of KBG syndrome.

A de novo pathogenic BMP2 variant-related phenotype with the novel finding of bicuspid aortic valve.

A rare autosomal dominant syndrome with craniofacial dysmorphisms, skeletal abnormalities, short stature, and congenital heart defects has recently been described, associated with monoallelic truncating and frameshift bone morphogenetic protein 2 (BMP2) variants and deletions. We describe a patient harboring a novel de novo BMP2 nonsense variant, who exhibited craniofacial and skeletal features previously described for this trait and the novel findings of bicuspid aortic valve (BAV) and aortic root and ascending aortic aneurysm. This first instance of aortic valve involvement provides another potential cause of BAV and confirms the role of BMP2 in left ventricular outflow development.

A rare cause of syndromic short stature: 3M syndrome in three families.

3M syndrome is a rare autosomal recessive genetic disorder characterized by severe growth retardation, dysmorphic facial features, skeletal dysplasia, and normal intelligence. Variants in CUL7, OBSL1, and CCDC8 genes have been reported to be responsible for this syndrome. In this study, the clinical and molecular findings of four 3M syndrome cases from three families are presented. All cases had growth retardation, relative macrocephaly, and typical dysmorphic facial features. Their neurological developments were normal. Sequencing of CUL7, OBSL1, and CCDC8 genes revealed two different novel homozygous variants in CUL7 in Families 1 and 3 and a previously reported homozygous pathogenic variant in OBSL1 in Family 2. In conclusion, a comprehensive dysmorphological evaluation should be obtained in individuals presenting with short stature and in such individuals with typical facial and skeletal findings, 3M syndrome should be considered. Our report expands the genotype of 3M syndrome and emphasizes the importance of thorough physical and dysmorphological examination.

A novel truncating variant in the FGD1 gene associated with Aarskog-Scott syndrome in a family previously diagnosed with Tel Hashomer camptodactyly.

Tel Hashomer camptodactyly syndrome is a long-known entity characterized by camptodactyly with muscular hypoplasia, skeletal dysplasia, and abnormal palmar creases. Currently, the genetic basis for this disorder is unknown, thus there is a possibility that this clinical presentation may be contained within another genetic diagnosis. Here, we present a multiplex family with a previous clinical diagnosis of Tel Hashomer camptodactyly syndrome. Whole exome sequencing and pedigree-based analysis revealed a novel hemizygous truncating variant c.269_270dup (p.Phe91Alafs*34) in the FGD1 gene (NM_004463.3) in all three symptomatic patients, congruous with a diagnosis of Aarskog-Scott syndrome. Our report adds to the limited data on Aarskog-Scott syndrome, and emphasizes the importance of unbiased comprehensive molecular testing toward establishing a diagnosis for genetic syndromes with unknown genetic basis.

Whole genome sequencing identifies pathogenic RNU4ATAC variants in a child with recurrent encephalitis, microcephaly, and normal stature.

Biallelic pathogenic variants in RNU4ATAC have been linked to microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1). Although children with MOPD1 have been reported to show profound, life-limiting clinical decompensation at the time of a febrile illness, these episodes including magnetic resonance imaging (MRI) findings have not been well characterized. We present acute MRI brain findings for a 10-year-old girl with homozygous variants in RNU4ATAC (NR_023343.1) n.55G>A, who presented with two episodes of clinical decompensation associated with a febrile illness in early childhood. The pathogenic variants were identified by whole genome sequencing as RNU4ATAC is not captured in most exome products. Her MRI of the brain revealed symmetric, diffusion restriction of the deep gray nuclei that initially pointed to a mitochondrial disease or acute necrotizing encephalopathy. Her phenotype included microcephaly and profound cognitive impairment that can be seen with MOPD1. However, she did not demonstrate clinical or radiographic evidence of a spondyloepimetaphyseal dysplasia or "primordial dwarfism" that is characteristic of this disease. As such, the predominant neurological presentation of this child represents an atypical variant of RNU4ATAC-associated disease and should be a diagnostic consideration for geneticists and neurologists caring for children, particularly in the event of an acute clinical decline.

Earlier detection of hypochondroplasia: A large single-center UK case series and systematic review.

Hypochondroplasia (HCH) is a rare autosomal dominant skeletal dysplasia condition caused by FGFR3 mutations leading to disproportionate short stature. Classically HCH presents in toddlers or school-age children, as limb-to-trunk disproportion and is often mild and easily overlooked during infancy. We report experiences from a single-center UK HCH-cohort of 31 patients, the rate of antenatal HCH detection in our cohort (13/31, 41.9%) and describe relevant case-data for this subset of 13 patients. Inclusion criteria were patients with confirmed molecular HCH diagnosis (by age 3 years) and presenting with short long-bones or large head size on antenatal ultrasound scan. We then conducted a systematic literature review using PUBMED and MEDLINE, analyzing patients with HCH and related antenatal findings. Antenatally suspected (with subsequent molecular confirmation) HCH has been reported 15 times in the literature (2004-2019). Key markers (consistent in both groups) included reduced; femur length, humeral length and increased; biparietal diameter and head circumference. HCH is increasingly detected both antenatally and in infancy, contrary to previous descriptions. This is likely due to greater HCH awareness, improved imaging, and easier molecular testing. Thus, one should consider HCH outside the classical presenting period. Studying the natural history of younger patients with HCH is important with the advent of several targeted FGFR3 therapies currently in trials for Achondroplasia, that may soon be trialed in HCH.

Clinical and molecular characterization of four patients with Robinow syndrome from different families.

Robinow syndrome (RS) is a rare heterogeneous disorder characterized by short stature, short-limbs, craniofacial, oro-dental abnormalities, vertebral segmentation defects, and frequently genital hypoplasia. Both autosomal dominant and recessive patterns of inheritance are observed with many causative genes. Here, we present the phenotypes and genotypes of four children with RS from different Indian families. Sequence variants were identified in genes ROR2, DVL1, and DVL3. Our results expand the mutational spectrum of RS and we also highlight the radiological changes in the radius and ulna in patients with ROR2 sequence variants which are primarily characteristic for ROR2 related RS but have been reported in WNT5A related RS.