Mutations in CDC45, Encoding an Essential Component of the Pre-initiation Complex, Cause Meier-Gorlin Syndrome and Craniosynostosis.

DNA replication precisely duplicates the genome to ensure stable inheritance of genetic information. Impaired licensing of origins of replication during the G1 phase of the cell cycle has been implicated in Meier-Gorlin syndrome (MGS), a disorder defined by the triad of short stature, microtia, and a/hypoplastic patellae. Biallelic partial loss-of-function mutations in multiple components of the pre-replication complex (preRC; ORC1, ORC4, ORC6, CDT1, or CDC6) as well as de novo stabilizing mutations in the licensing inhibitor, GMNN, cause MGS. Here we report the identification of mutations in CDC45 in 15 affected individuals from 12 families with MGS and/or craniosynostosis. CDC45 encodes a component of both the pre-initiation (preIC) and CMG helicase complexes, required for initiation of DNA replication origin firing and ongoing DNA synthesis during S-phase itself, respectively, and hence is functionally distinct from previously identified MGS-associated genes. The phenotypes of affected individuals range from syndromic coronal craniosynostosis to severe growth restriction, fulfilling diagnostic criteria for Meier-Gorlin syndrome. All mutations identified were biallelic and included synonymous mutations altering splicing of physiological CDC45 transcripts, as well as amino acid substitutions expected to result in partial loss of function. Functionally, mutations reduce levels of full-length transcripts and protein in subject cells, consistent with partial loss of CDC45 function and a predicted limited rate of DNA replication and cell proliferation. Our findings therefore implicate the preIC as an additional protein complex involved in the etiology of MGS and connect the core cellular machinery of genome replication with growth, chondrogenesis, and cranial suture homeostasis.

Maladaptative Autophagy Impairs Adipose Function in Congenital Generalized Lipodystrophy due to Cavin-1 Deficiency.

CONTEXT: Mutations in PTRF encoding cavin-1 are responsible for congenital generalized lipodystrophy type 4 (CGL4) characterized by lipoatrophy, insulin resistance, dyslipidemia, and muscular dystrophy. Cavin-1 cooperates with caveolins to form the plasma membrane caveolae, which are involved in cellular trafficking and signalling and in lipid turnover. OBJECTIVE: We sought to identify PTRF mutations in patients with CGL and to determine their impact on insulin sensitivity, adipose differentiation, and cellular autophagy. DESIGN AND PATIENTS: We performed phenotyping studies and molecular screening of PTRF in two unrelated families with CGL. Cellular studies were conducted in cultured skin fibroblasts from the two probands and from control subjects, and in murine 3T3-F442A preadipocytes. Knockdown of cavin-1 or ATG5 was obtained by small interfering RNA-mediated silencing. RESULTS: We identified two new PTRF homozygous mutations (p.Asp59Val or p.Gln157Hisfs*52) in four patients with CGL4 presenting with generalized lipoatrophy and associated metabolic abnormalities. In probands' fibroblasts, cavin-1 expression was undetectable and caveolin-1 and -2 barely expressed. Ultrastructural analysis revealed a loss of membrane caveolae and the presence of numerous cytoplasmic autophagosomes. Patients' cells also showed increased autophagic flux and blunted insulin signaling. These results were reproduced by PTRF knockdown in control fibroblasts and in 3T3-F442A preadipocytes. Cavin-1 deficiency also impaired 3T3-F442A adipocyte differentiation. Suppression of autophagy by small interfering RNA-mediated silencing of ATG5 improved insulin sensitivity and adipocyte differentiation. CONCLUSIONS: This study showed that cavin-1 deficiency resulted in maladaptative autophagy that contributed to insulin resistance and altered adipocyte differentiation. These new pathophysiological mechanisms could open new therapeutic perspectives for adipose tissue diseases including CGL4.

A case of infantile osteopetrosis: The radioclinical features with literature update.

BACKGROUND: Osteopetrosis is a rare hereditary metabolic bone disorder characterized by generalized skeletal sclerosis caused by a defect in bone resorption and remodelling. Infantile autosomal recessive osteopetrosis is one of three subtypes of osteopetrosis and the most severe form. The correct and early diagnosis of infantile osteopetrosis is important for management of complications and for future genetic counselling. Diagnosis is largely based on clinical and radiographic evaluation, confirmed by gene testing where applicable. METHODS: Therefore, in this case study the classical clinical and radiological signs of a boy with infantile osteopetrosis will be presented with a comprehensive literature update. The differentiating signs from other causes of hereditary osteosclerosing dysplasias are discussed. RESULTS: This case study and review of available literature show that there tends to be a highly unique clinical and skeletal radiographic pattern of affection in infantile osteopetrosis. CONCLUSION: Although tremendous advances have been made in the elucidation of the genetic defect of osteopetrosis over the past years, the role of accurate clinical and radiological assessment remains an important contributor to the diagnosis of infantile osteopetrosis.

Non-manifesting AHI1 truncations indicate localized loss-of-function tolerance in a severe Mendelian disease gene.

Determination of variant pathogenicity represents a major challenge in the era of high-throughput sequencing. Erroneous categorization may result if variants affect genes that are in fact dispensable. We demonstrate that this also applies to rare, apparently unambiguous truncating mutations of an established disease gene. By whole-exome sequencing (WES) in a consanguineous family with congenital non-syndromic deafness, we unexpectedly identified a homozygous nonsense variant, p.Arg1066*, in AHI1, a gene associated with Joubert syndrome (JBTS), a severe recessive ciliopathy. None of four homozygotes expressed any signs of JBTS, and one of them had normal hearing, which also ruled out p.Arg1066* as the cause of deafness. Homozygosity mapping and WES in the only other reported JBTS family with a homozygous C-terminal truncation (p.Trp1088Leufs*16) confirmed AHI1 as disease gene, but based on a more N-terminal missense mutation impairing WD40-repeat formation. Morpholinos against N-terminal zebrafish Ahi1, orthologous to where human mutations cluster, produced a ciliopathy, but targeting near human p.Arg1066 and p.Trp1088 did not. Most AHI1 mutations in JBTS patients result in truncated protein lacking WD40-repeats and the SH3 domain; disease was hitherto attributed to loss of these protein interaction modules. Our findings indicate that normal development does not require the C-terminal SH3 domain. This has far-reaching implications, considering that variants like p.Glu984* identified by preconception screening ('Kingsmore panel') do not necessarily indicate JBTS carriership. Genomes of individuals with consanguineous background are enriched for homozygous variants that may unmask dispensable regions of disease genes and unrecognized false positives in diagnostic large-scale sequencing and preconception carrier screening.

Autosomal dominant SCA5 and autosomal recessive infantile SCA are allelic conditions resulting from SPTBN2 mutations.

Although many genes have been identified for the autosomal recessive cerebellar ataxias (ARCAs), several patients are unlinked to the respective loci, suggesting further genetic heterogeneity. We combined homozygosity mapping and exome sequencing in a consanguineous Egyptian family with congenital ARCA, mental retardation and pyramidal signs. A homozygous 5-bp deletion in SPTBN2, the gene whose in-frame mutations cause autosomal dominant spinocerebellar ataxia type 5, was shown to segregate with ataxia in the family. Our findings are compatible with the concept of truncating SPTBN2 mutations acting recessively, which is supported by disease expression in homozygous, but not heterozygous, knockout mice, ataxia in Beagle dogs with a homozygous frameshift mutation and, very recently, a homozygous SPTBN2 nonsense mutation underlying infantile ataxia and psychomotor delay in a human family. As there was no evidence for mutations in 23 additional consanguineous families, SPTBN2-related ARCA is probably rare.

WNT1 mutations in families affected by moderately severe and progressive recessive osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a heritable disorder that ranges in severity from death in the perinatal period to an increased lifetime risk of fracture. Mutations in COL1A1 and COL1A2, which encode the chains of type I procollagen, result in dominant forms of OI, and mutations in several other genes result in recessive forms of OI. Here, we describe four recessive-OI-affected families in which we identified causative mutations in wingless-type MMTV integration site family 1 (WNT1). In family 1, we identified a homozygous missense mutation by exome sequencing. In family 2, we identified a homozygous nonsense mutation predicted to produce truncated WNT1. In family 3, we found a nonsense mutation and a single-nucleotide duplication on different alleles, and in family 4, we found a homozygous 14 bp deletion. The mutations in families 3 and 4 are predicted to result in nonsense-mediated mRNA decay and the absence of WNT1. WNT1 is a secreted signaling protein that binds the frizzled receptor (FZD) and the coreceptor low-density lipoprotein-receptor-related protein 5 (LRP5). Biallelic loss-of-function mutations in LRP5 result in recessive osteoporosis-pseudoglioma syndrome with low bone mass, whereas heterozygous gain-of-function mutations result in van Buchem disease with elevated bone density. Biallelic loss-of-function mutations in WNT1 result in a recessive clinical picture that includes bone fragility with a moderately severe and progressive presentation that is not easily distinguished from dominant OI type III.

Further evidence of the clinical, hormonal, and genetic heterogeneity of Klinefelter syndrome: a study of 216 infertile Egyptian patients.

This study aims to provide further insight into the phenotypic heterogeneity of Klinefelter syndrome (KS) by presenting clinical, hormonal, and genetic data from a large series of Egyptian infertile patients with KS. A retrospective case series of KS patients was studied over a period from January 2003 to April 2010. All patients underwent a complete history and physical examination; color duplex examination; semen analysis; measurement of total testosterone (T), follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), and prolactin (PRL); and chromosomal typing. Mosaic KS diagnosis was confirmed by fluorescence in situ hybridization. The series included 216 KS patients (198 nonmosaic, 16 mosaic, and 2 KS variants). Typical clinical signs of hypoandrogenism were observed in 86% of patients. Gynecomastia affected 20.8% of the patients. Eunuchoidal body proportions, with arm span exceeding height and lower segment length exceeding upper segment length, were detected in 43.9% and 64.4% of the patients, respectively. In all patients, a reduction in testicular size and azoospermia were detected. Normal levels of T, FSH, LH, E2, and PRL were detected in 44.5%, 3.7%, 3.3%, 93.5%, and 91.2% of patients, respectively. Differences were not significant between patients with classic KS and those with mosaic KS in terms of the frequency of clinical signs of hypoandrogenism, gynecomastia, low T concentrations, or high concentrations of FSH, LH, E2, and PRL (all P > .05). The results of the current study emphasize the heterogeneous clinical, hormonal, and genetic phenotype of infertile KS patients. Our findings support the usefulness of cytogenetic studies in infertile patients showing small testicular size and azoospermia, regardless of the presence of other clinical or endocrine findings.

Mutations in KIF7 link Joubert syndrome with Sonic Hedgehog signaling and microtubule dynamics.

Joubert syndrome (JBTS) is characterized by a specific brain malformation with various additional pathologies. It results from mutations in any one of at least 10 different genes, including NPHP1, which encodes nephrocystin-1. JBTS has been linked to dysfunction of primary cilia, since the gene products known to be associated with the disorder localize to this evolutionarily ancient organelle. Here we report the identification of a disease locus, JBTS12, with mutations in the KIF7 gene, an ortholog of the Drosophila kinesin Costal2, in a consanguineous JBTS family and subsequently in other JBTS patients. Interestingly, KIF7 is a known regulator of Hedgehog signaling and a putative ciliary motor protein. We found that KIF7 co-precipitated with nephrocystin-1. Further, knockdown of KIF7 expression in cell lines caused defects in cilia formation and induced abnormal centrosomal duplication and fragmentation of the Golgi network. These cellular phenotypes likely resulted from abnormal tubulin acetylation and microtubular dynamics. Thus, we suggest that modified microtubule stability and growth direction caused by loss of KIF7 function may be an underlying disease mechanism contributing to JBTS.

Phenotypic and genetic characterization of a cohort of pediatric Wilson disease patients.

BACKGROUND: In Egypt, Wilson disease seems to be under diagnosed and clinical data on large cohorts are limited. The aim of this study is to highlight the clinical, laboratory and genetic characteristics of this disease in our pediatric population as well as to report our experience with both treatment options and outcome. METHODS: The study included 77 patients from 50 unrelated families (62 were followed up for a mean period of 58.9 ± 6.4 months and 27 were asymptomatic siblings). Data were collected retrospectively by record analysis and patient interviews. Diagnosis was confirmed by sequencing of the ATP7B gene in 64 patients. RESULTS: Our patients had unique characteristics compared to other populations. They had a younger age of onset (median: 10 years), higher prevalence of Kayser-Fleischer rings (97.6% in the symptomatic patients), low ceruloplasmin (93.5%), high rate of parental consanguinity (78.9%) as well as a more severe course. 71.42% of those on long term D-penicillamine improved or were stable during the follow up with severe side effects occurring in only 11.5%. Preemptive treatment with zinc monotherapy was an effective non-toxic alternative to D-penicillamine. Homozygous mutations were found in 85.7%, yet limited by the large number of mutations detected, it was difficult to find genotype-phenotype correlations. Missense mutations were the most common while protein-truncating mutations resulted in a more severe course with higher incidence of acute liver failure and neurological symptoms. CONCLUSIONS: Egyptian children with Wilson disease present with early Kayser-Fleischer rings and early onset of liver and neurological disease. The mutational spectrum identified differs from that observed in other countries. The high rate of homozygous mutations (reflecting the high rate of consanguinity) may potentially offer further insights on genotype-phenotype correlation.

Cholestasis in patients with Cockayne syndrome and suggested modified criteria for clinical diagnosis.

BACKGROUND: Cockayne syndrome is a rare autosomal recessive neurodegenerative disease characterized by low-to-normal birth weight; growth failure; brain dysmyelination with calcium deposits, cutaneous photosensitivity; pigmentary retinopathy, cataract, and sensorineural hearing loss. To the best of our knowledge, cholestatic liver disease was not previously reported in these patients. AIM: To highlight the presence of cholestasis and liver dysfunction in this group of patients and to suggest modified criteria for clinical diagnosis. METHODS: The study included nine patients with Cockayne from four different families (five males and four females) in which Cockayne was suspected clinically. In all patients chromosomal breakage studies revealed mild (45%) to moderate (60%) increase in frequency of chromatid and chromosome gaps and breaks versus 25% in normal controls. Diagnosis was confirmed by DNA repair assay. RESULTS: During routine follow up of these patients, seven of them had evident liver affection ranging from mild elevation in liver enzymes to cholestatic liver disease and liver cell failure. The attacks were recurrent in two patients and were sometimes preceded by infection. The attack may lead to deterioration of neurological and/or liver condition. It may end in liver cell failure that either recovers completely or may lead to death. CONCLUSIONS: liver disease could be considered common in Egyptian patients with Cockayne with the cholestatic form being the most evident. The syndrome should be included in the list of causes of cholestatic liver disease. Chromosomal breakage study and positive family history should be included as major criteria for clinical diagnosis of Cockayne especially in a population like ours where consanguineous marriage is very high and molecular testing and UV sensitivity tests are considered unaffordable.

Factor V G1691A (Leiden) is a major etiological factor in Egyptian Budd-Chiari syndrome patients.

OBJECTIVE: Budd-Chiari syndrome is a multifactorial disease in which several prothrombotic disorders may predispose patients to the development of thrombosis at this uncommon location (hepatic veins). The aim of this study was to determine the prevalence and characteristics of inherited thrombophilia in Egyptian Budd-Chiari syndrome patients. METHODS: The study included 47 Budd-Chiari syndrome patients (20 children and 27 adults). Genotyping of Factor V G1691A (Leiden), prothrombin G20210A (PT), and methylenetetrahydrofolate reductase C677T were performed using real-time PCR and fluorescence melting curve detection analysis. RESULTS: Factor V Leiden was observed in 29 patients (61.7%). It is the only factor that caused Budd-Chiari syndrome in 18 of the patients and in 5 of the patients with inferior vena cava involvement. Myeloproliferative disease was noted in 12 (25.5%) patients, antiphospholipid syndrome in 5 (10.6%), and Behcet's disease in 3 (6.4%). Interestingly, 3 of the children with Budd-Chiari syndrome had lipid storage disease. CONCLUSION: Factor V Leiden was a major etiological factor in Egyptian Budd-Chiari syndrome patients, which may have been related to the high frequency of this mutation in the study region. Factor V Leiden was also a strong thrombophilic factor and the leading cause of inferior vena cava thrombosis in these patients. Lipid storage disease should be included as a risk factor for Budd-Chiari syndrome.

Cytogenetic aberrations and the development of uterine leiomyomata.

This study was conducted to define the cytogenetically critical regions of uterine leiomyomata, hoping to demonstrate the presence of possible genes involved in their evolution. It was carried out on 25 randomly selected uterine leiomyoma specimens obtained from 16 patients during hysterectomy or myomectomy operations. Successful tissue culture and karyotyping were performed in 19 specimens. There was no correlation between patient age, gravidity, or presenting symptom and the presence of chromosomal abnormality. A significant correlation was found between short culture turnaround time and the occurrence of chromosomal abnormality. Abnormal clonal karyotypes were present in 6 specimens, non-clonal abnormalities in 4 specimens and normal karyotypes were found in 9 specimens. Myomas with cross section >4 cm showed an increased incidence of abnormal karyotypes and a statistically significant higher incidence of clonal abnormalities. On the other hand, submucous myomas presented fewer clonal abnormalities than did intramural or subserosal myomas. Clonal chromosomal abnormalities involved 5 different chromosomes (2, 7, 8, 12, 22), which indicate genetic heterogeneity of such benign tumors and the need of molecular cytogenetic studies or molecular studies to characterize possible candidate genes at specific chromosomal breakpoints.

Prognostic value of the t(14;18)(q32;q21) in patients with diffuse large B-cell lymphoma.

The prognostic significance of the t(14;18) in diffuse large B-cell lymphoma is still controversial. To assess the impact of the t(14;18) on patient survival, we investigated 26 patients with diffuse large B-cell lymphoma for the presence of t(14;18). The t(14;18) was detected in 90.9% of patients with high international prognostic index score. The five-year overall survival was 0.0% and 68.75% in positive and negative cases of t(14;18) respectively. The detection of the t(14;18) combined with the international prognostic index score is a useful strategy for more appropriate risk stratification and prediction of outcome of patients with diffuse large B-cell lymphoma.

Mutational analysis of ATP7B gene in Egyptian children with Wilson disease: 12 novel mutations.

The aim of this work was to study the mutations within ATP7B in Egyptian children with Wilson disease and to evaluate any potential correlation between genotype and phenotype in this cohort. The study consisted of 48 children with Wilson disease from 32 independent families. The 21 exons of the ATP7B gene were amplified in a thermal cycler. Direct sequencing of the amplified polymerase chain reaction (PCR) products was performed by cycle sequencing using fluorescent dye terminators in an automatic ABI sequencer. Thirty-one different mutations in 96 chromosomes were detected (19 missense, three nonsense, seven frameshift deletions, and two splice-site mutations). Of these, 12 mutations have not been previously reported. The p.N1270S, p.C703Y, IVS18-2A > G, p.R1319X, c.2304-2305insC, and p.H1069Q were present in 7.8%, 6.2%, 6.2%, 6.2%, 4.7%, and 4.7%, respectively, of studied chromosomes in independent families. One patient was homozygous for both p.N1270S and p.T1434M mutations. Frameshift and nonsense mutations were found in 50% of patients with disease onset < or =8 years compared with only 26% in patients with onset >8 years. Despite mutation heterogeneity in Egyptian children, genotype-phenotype correlation analysis seems to be promising in this population, as many patients carry homozygous mutations, a situation that mandates a larger-scale population screening to identify the carrier rate in this community.

Rapid sex determination using PCR technique compared to classic cytogenetics.

BACKGROUND: Fetal sexual differentiation relies on the translation of chromosomal sex established at fertilization into gonadal sex and somatic sex as development proceeds. In cases where chromosomal, gonadal, and somatic sex are incongruent in human infants and children, rapid establishment of the diagnosis and implementation of medical and surgical management is of paramount importance, since the gender identity is so important to the psychological well-being throughout life. OBJECTIVE: This work was done in order to test the value of PCR technique for rapid sex determination compared to classic cytogenetic technique. METHODS: Subjects included 20, cases including 10 neonates with ambiguous genitalia, 2 adult females with delayed puberty and 8 adult males with infertility, in addition to 20 normal infants of both sexes as a control group. The diagnosis of sex was attempted through examination, cytogenetic study, ultrasonography, gonadal biopsy and hormonal analysis, in addition to PCR amplification for the detection of SRY and ATL1 gene loci on Y and X chromosomes respectively. RESULTS: Four neonates were diagnosed as partial testicular feminization showed both positive bands for the Y and X chromosomes and a karyogram of 46/XY. Three neonates were diagnosed as true hermaphrodites showed positive amplification for both Y and X chromosomes with a mosaic karyogram 46,XX/XY. Three neonates were diagnosed as cases of adrenogenital syndrome showed positive amplification of only the Xchromosome and had a karyogram of 46/XX. One of the two adult females was diagnosed as turner syndrome showed positive amplification of the X chromosome and a karyogram of 45/XO; the other one was diagnosed as complete testicular feminization had a positive amplification of X and Y chromosomes and a karyogram of 46/XY. The 8 adult males with infertility showed a positive amplification of X and Y chromosome and a karyogram of 47/XXY (Klinefelter syndrome) in 7 cases and 46/XY gonadal dysgenesis in one case. CONCLUSION: We concluded that PCR as a simple, rapid and reliable technique can complement and also confirm cytogenetic studies in the diagnosis of sex in cases of sex chromosome disorders.

Frequency of five thrombophilic polymorphisms in the Egyptian population.

This study was conducted to evaluate the frequency of FV1691 G-A, FV4070 A-G, PT20210 G-A, EPCR 23 gene bp insertion and ACE gene 300 bp deletion among healthy Egyptians. One hundred and eighty eight healthy Egyptians were included to the study. Previously reported molecular techniques were used for the determination of mutations. Thirthy one individuals had FV1691 G-A (16.5%) mutation with an allele frequency of 0.09. R2 and R3 haplotypes were found in 21 (11.2%) and 4 (2.1%) individuals, respectively. Only one healthy individual had EPCR gene 23 bp insertion (0.53%) and two individuals (1.06%) had PT20210 G-A mutation, respectively. The ACE gene -300 bp del in homozygous state was present in 92 (48.9%) individuals. The frequency of D allele was 0.718. Our preliminary data revealed that FV1691 G-A mutation is very frequent among Egyptians and it will be meaningfull to study the mutation also in the thrombotic events in Egypt.