The 12q14 microdeletion syndrome: six new cases confirming the role of HMGA2 in growth

We report six patients with array deletions encompassing 12q14. Out of a total of 2538 array investigations carried out on children with developmental delay and dysmorphism in three diagnostic testing centres, six positive cases yielded a frequency of 1 in 423 for this deletion syndrome. The deleted region in each of the six cases overlaps significantly with previously reported cases with microdeletions of this region. The chromosomal range of the deletions extends from 12q13.3q15. In the current study, we report overlapping deletions of variable extent and size but primarily comprising chromosomal bands 12q13.3q14.1. Four of the six deletions were confirmed as de novo events. Two cases had deletions that included HMGA2, and both children had significant short stature. Neither case had osteopoikilosis despite both being deleted for LEMD3. Four cases had deletions that ended proximal to HMGA2 and all of these had much better growth. Five cases had congenital heart defects, including two with atrial septal defects, one each with pulmonary stenosis, sub-aortic stenosis and a patent ductus. Four cases had moderate delay, two had severe developmental delay and a further two had a diagnosis of autism. All six cases had significant speech delay with subtle facial dysmorphism.     

A Novel Homozygous Nonsense Mutation in the PVRL4 Gene and Expansion of Clinical Spectrum of EDSS1

Ectodermal dysplasias (EDs) belong to a group of genetic diseases which result from alterations in ectoderm‐derived appendages including hair, nail, teeth and sweat glands. Ectodermal dysplasia syndactyly syndrome (EDSS1) is one of the rare forms of ED caused by mutations in nectin‐4, encoded by the PVRL4 gene. The present study described clinical investigation of the EDSS1 identified in a large consanguineous family. Furthermore, DNA sequence analysis revealed a novel homozygous nonsense mutation (181C>T, p.Asp61*) in the PVRL4 gene.     

IL‐12Rβ1 Deficiency: Mutation Update and Description of the IL12RB1 Variation Database

IL‐12Rβ1 deficiency is an autosomal recessive disorder characterized by predisposition to recurrent and/or severe infections caused by otherwise poorly pathogenic mycobacteria and salmonella. IL‐12Rβ1 is a receptor chain of both the IL‐12 and the IL‐23 receptor and deficiency of IL‐12Rβ1 thus abolishes both IL‐12 and IL‐23 signaling. IL‐12Rβ1 deficiency is caused by bi‐allelic mutations in the IL12RB1 gene. Mutations resulting in premature stop codons, such as nonsense, frame shift, and splice site mutations, represent the majority of IL‐12Rβ1 deficiency causing mutations (66%; 46/70). Also every other morbid mutation completely inactivates the IL‐12Rβ1 protein. In addition to disease‐causing mutations, rare and common variations with unknown functional effect have been reported in IL12RB1. All these variants have been deposited in the online IL12RB1 variation database ( www.LOVD.nl/IL12RB1 ). In this article, we review the function of IL‐12Rβ1 and molecular genetics of human IL12RB1.     

Novel Mutations in the DYNC1H1 Tail Domain Refine the Genetic and Clinical Spectrum of Dyneinopathies

The heavy chain 1 of cytoplasmic dynein (DYNC1H1) is responsible for movement of the motor complex along microtubules and recruitment of dynein components. Mutations in DYNC1H1 are associated with spinal muscular atrophy (SMA), hereditary motor and sensory neuropathy (HMSN), cortical malformations, or a combination of these. Combining linkage analysis and whole‐exome sequencing, we identified a novel dominant defect in the DYNC1H1 tail domain (c.1792C>T, p.Arg598Cys) causing axonal HMSN. Mutation analysis of the tail region in 355 patients identified a de novo mutation (c.791G>T, p.Arg264Leu) in an isolated SMA patient. Her phenotype was more severe than previously described, characterized by multiple congenital contractures and delayed motor milestones, without brain malformations. The mutations in DYNC1H1 increase the interaction with its adaptor BICD2. This relates to previous studies on BICD2 mutations causing a highly similar phenotype. Our findings broaden the genetic heterogeneity and refine the clinical spectrum of DYNC1H1, and have implications for molecular diagnostics of motor neuron diseases.     

Untreated growth hormone deficiency with extremely short stature, bone dysplasia, cleft lip--palate and severe mental retardation in a 26-year-old man with a de novo unbalanced translocation t(1;12)(q24;q24)

We report on a 26-year-old patient presenting with extremely short stature (height 72 cm, weight 6.5 kg, OFC 42.5 cm), facial dysmorphism, cleft lip–palate, severe mental retardation and de novo 1q24.2–q25.2 and 12q24.31 interstitial deletion. He was the only child of non-consanguineous parents and his birth length was 43 cm. He had severe feeding difficulties and required enteral nutrition until the age of 3 years. Standard cytogenetic analysis showed an apparently balanced de novo translocation t(1;12)(q24;q24). Endocrine studies at 11 years of age for severe growth retardation revealed multiple pituitary hormone deficiency with severe growth hormone deficiency, but the child was untreated because of associated mental retardation. At 26 years of age, he could not walk or speak and had no signs of puberty. Investigations revealed spondylo-epi-metaphyseal dysplasia with severe osteoporosis, enlarged aorta when compared to the patient's size and apparently normal pituitary development. High resolution karyotype showed a 1q24–q25 deletion, and comparative genomic hybridization studies confirmed the 1q interstitial deletion. FISH studies of both breakpoints using PACs and BACs enabled us to further characterize the 1q interstitial deletion (1q24.2–1q25.2) and also revealed a 12q24.31 interstitial microdeletion. This case is compared with previously reported patients with similar deletions, but the untreated pituitary deficiency could also be responsible in part for the severity of the growth deficiency. This observation is of interest for two reasons. First, these deletions could be a clue in the search for a gene responsible for growth hormone deficiency/midline defects. Second, it shows the importance of molecular cytogenetics in the study of de novo apparently balanced translocation with abnormal phenotype.     

特发性矮小症儿童血清GH、IGF-1、微量元素与体格发育指标的相关性分析

目的调查分析特发矮小症(ISS)儿童血清中生长激素(GH)、胰岛素生长因子-1(IGF-1)水平及钙镁锌等微量元素状况。方法选取2014年2月至2018年2月我院收治的矮小症儿童230例为研究对象(其中138例ISS患儿纳入ISS组,余下92例非ISS矮小症儿童纳入非ISS组),另选择同期体检的健康儿童50例为对照组,采用精氨酸激发试验及可乐定激发试验检测其血清GH水平,并分析三组IGF-1、微量元素(钙镁锌、维生素D)水平、体格发育指标[身高、体重、体质量指数(BMI)、瘦素(Leptin)、骨钙素(Ost)],分析ISS儿童GH、IGF-1水平与微量元素水平、体格发育指标的相关性。结果ISS组可乐定、精氨酸激发试验阳性率高于非ISS组(P<0.05);ISS组、非ISS组血清GH、IGF-1、钙、镁、锌及维生素D水平低于对照组,ISS组血清GH、IGF-1、钙及维生素D水平低于非ISS组(P<0.05);ISS组、非ISS组的身高、体重、BMI及血清Ost水平低于对照组,而Leptin水平高于对照组,ISS组的身高、体重及血清Leptin、Ost水平也低于非ISS组(P<0.05);Spearman相关性分析发现,ISS儿童血清GH、IGF-1水平与血清钙、锌、维生素D、BMI、Ost呈正相关,与Leptin呈负相关(P<0.05),而矮小症儿童血清GH、IGF-1水平与血清钙、镁、维生素D、BMI呈正相关,与Leptin呈负相关(P<0.05)。结论ISS儿童血清GH、IGF-1水平普遍偏低,且其钙镁锌等微量元素明显不足,可通过GH/IGF-1轴、微量元素对其进行诊断,并合理补充微量元素。     

NPHS2 Mutations in Steroid‐Resistant Nephrotic Syndrome: A Mutation Update and the Associated Phenotypic Spectrum

Mutations in the NPHS2 gene encoding podocin are implicated in an autosomal‐recessive form of nonsyndromic steroid‐resistant nephrotic syndrome in both pediatric and adult patients. Patients with homozygous or compound heterozygous mutations commonly present with steroid‐resistant nephrotic syndrome before the age of 6 years and rapidly progress to end‐stage kidney disease with a very low prevalence of recurrence after renal transplantation. Here, we reviewed all the NPHS2 mutations published between October 1999 and September 2013, and also all novel mutations identified in our personal cohort and in international genetic laboratories. We identified 25 novel pathogenic mutations in addition to the 101 already described. The mutations are distributed along the entire coding region and lead to all kinds of alterations including 53 missense, 17 nonsense, 11 small insertions, 26 small deletions, 16 splicing, two indel mutations, and one mutation in the stop codon. In addition, 43 variants were classified as variants of unknown significance, as these missense changes were exclusively described in the heterozygous state and/or considered benign by prediction software. Genotype–phenotype analyses established correlations between specific variants and age at onset, ethnicity, or clinical evolution. We created a Web database using the Leiden Open Variation Database ( www.lovd.nl/NPHS2 ) software that will allow the inclusion of future reports.     

Mutational Spectrum of the C1 Inhibitor Gene in a Cohort of Italian Patients with Hereditary Angioedema: Description of Nine Novel Mutations

Hereditary angioedema (HAE) is an autosomal dominant disease due to mutations in the C1 inhibitor gene (C1NH) that affects protein synthesis (HAE type I) or function (HAE type II). In 45 subjects affected by HAE diagnosed through clinical features and C1 inhibitor deficiency from the south of Italy (38 with type I and 7 with type II HAE), the whole C1NH coding region was screened for mutations by direct DNA sequencing. A severity score based on clinical manifestation, age at disease onset, and need for long‐term prophylaxis was used to investigate possible genotype‐phenotype correlations. A series of 22 different mutations was identified: nine missense (40.9%), five nonsense (22.7%), six frameshift (27.3), one small deletion (4.5%), and one splicing defect (4.5%). Nine C1NH mutations have not been previously described. No correlation was found between C1 inhibitor function level and severity score or age at first attack. Moreover, there was no correlation between different types of mutations and clinical phenotype. The number of different mutations identified highlights the heterogeneity of C1 inhibitor deficiency and supports the hypothesis that HAE clinical phenotype is not strictly related to the type of mutation but rather depends on unknown factors.     

IL-12对CIK细胞体外增殖及细胞毒活性影响的研究

目的观察IL-12对细胞因子诱导的杀伤细胞（CIK）体外扩增和细胞毒活性的影响。方法采用3种不同的细胞因子组合扩增CIK细胞,即IL-2组：IFN-1、CD3单抗、IL-1和IL-2;IL-2加IL-12组：IFN-γ、CD3单抗、IL-1、IL-2和IL-12;IL：12组：IFN-γ、CD3单抗、IL-1和IL-12。细胞计数法测定细胞的增殖、流式细胞术分析细胞表型,MTT法测定细胞毒作用。结果3种方法均可使细胞增殖能力显著增加,对CD3^＋CD56^＋细胞的诱导作用也无明显差异,IL-12与IL-2联合作用组促增殖能力和细胞毒性作用明显强于其他两组,P〈0．05,差异有统计学意义。结论IL-12同样可以用于CIK细胞的诱导,而联合IL-12与IL-2可以显著增强CIK细胞的增殖能力和细胞毒性。     

Expansion of restricted cellular immune responses to HIV-1 envelope by vaccination: IL-7 and IL-12 differentially augment cellular proliferative responses to HIV-1

The failure of immune effector mechanisms to control HIV-1 infection has important consequences for the human host. In a randomized cohort of HIV-infected patients, there was striking in vitro restriction of the proliferative response to HIV-1 envelope protein (Env), gp160; only 34% of patients recognized Env. Therapeutic vaccination with recombinant gp160 or gp120 (rgp160, rgp120) reversed the restriction in vitro, with Env recognition rising to 81%. Peripheral blood mononuclear cells (PBMC) from HIV-infected vaccine recipients, placebo recipients, and seronegative volunteers were cultured with exogenous IL-7 or IL-12 and either tetanus toxoid (TT) or gp160. IL-7 significantly augmented proliferative responses to TT and gp160, whereas IL-12 only affected proliferation to gp160. IL-7, but not IL-12, increased the number of HIV-infected placebo recipients who recognized rgp160. IL-12 had its greatest effect in the induction of rgp160-specific responses from seronegative individuals. The data suggest that these two cytokines have differential activity in the relief of restricted cellular immunity to Env; the predominant effect of IL-7 is in individuals who have been primed by exposure to antigen, while the effect of IL-12 is most evident in seronegative, unprimed individuals. Modification of restricted proliferative responses to Env by vaccination or cytokines in vitro suggests that strategies incorporating IL-7 or IL-12 as adjuvants may selectively boost cellular reactivity to HIV-1.     

Mutations of the Imprinted CDKN1C Gene as a Cause of the Overgrowth Beckwith–Wiedemann Syndrome: Clinical Spectrum and Functional Characterization

Beckwith–Wiedemann syndrome (BWS) is an imprinting disorder associating macroglossia, abdominal wall defects, visceromegaly, and a high risk of childhood tumor. Molecular anomalies are mostly epigenetic; however, mutations of CDKN1C are implicated in 8% of cases, including both sporadic and familial forms. We aimed to describe the phenotype of BWS patients with CDKN1C mutations and develop a functional test for CDKN1C mutations. For each propositus, we sequenced the three exons and intron–exon boundaries of CDKN1C in patients presenting a BWS phenotype, including abdominal wall defects, without 11p15 methylation defects. We developed a functional test based on flow cytometry. We identified 37 mutations in 38 pedigrees (50 patients and seven fetuses). Analysis of parental samples when available showed that all mutations tested but one was inherited from the mother. The four missense mutations led to a less severe phenotype (lower frequency of exomphalos) than the other 33 mutations. The following four tumors occurred: one neuroblastoma, one ganglioneuroblastoma, one melanoma, and one acute lymphoid leukemia. Cases of BWS caused by CDKN1C mutations are not rare. CDKN1C sequencing should be performed for BWS patients presenting with abdominal wall defects or cleft palate without 11p15 methylation defects or body asymmetry, or in familial cases of BWS.     

Effects of interleukin-12 administration during the pre- and peri-implantation period on mouse embryofetal development

PROBLEM: The immunological success of pregnancy is thought to depend upon the establishment of a balance between favorable and deleterious cytokines, the current paradigm viewing pregnancy as a T helper (Th)2 cytokine-dependent phenomenon. In this context, a particular attention should be directed to the potential role of interleukin (IL)-12, which promotes the development of Th1 responses, in the induction of adverse pregnancy-related phenomena. Indeed, very few data linked the Th1-inducer IL-12 to the event of abortion. METHODS: In this study, we have investigated the maternal and fetal effects of exogenous administration of IL-12 to CD1 (BR) ICR mice during the pre- and peri-implantation period (day 2-6 of pregnancy). Animals have been evaluated for parameters of reproductive performance, embryo and fetal developmental toxicity and maternal toxicity. RESULTS: Intraperitoneal administration of IL-12 at concentrations from 2.5 to 10 microg/kg daily did not result in an increase in the murine abortion rate. A statistically significant, although minimal, decrease in the number of somites were found in the embryos of animals treated with IL-12 at a dose of 10 microg/kg/day. However, developmental parameters at birth were similar between the two groups of animals suggesting that alteration of somites might be a transitory state during treatment. An increased body weight gains and reduced feed and water consumption were observed in the mothers treated with the cytokine. CONCLUSION: In the present experimental conditions and in this specific strain of mice, IL-12 does not exert adverse effects on reproductive performance and induces an only modest harmful action on mothers and embryos.     

Novel antibodies that selectively block mouse IL-12 enable the re-evaluation of the role of IL-12 in immune protection and pathology

The dimeric cytokine IL-12 is important in the control of various infections but also contributes to the pathology of certain diseases making it a potential target for therapy. However, its specific inhibition with antibodies is complicated by the fact that its two subunits are present in other cytokines: p40 in IL-23 and p35 in IL-35. This has led to erroneous conclusions like the alleged implication of IL-12 in experimental autoimmune encephalomyelitis (EAE). Here, we report the development of a mouse anti-mouse IL-12 vaccine and the production of monoclonal antibodies (mAbs) that do not react with p40 or p35 (in IL-35) but specifically recognize and functionally inhibit the IL-12 heterodimer. Using one of these mAbs, MM12A1.6, that strongly inhibited IFN-γ production and LPS-induced septic shock after viral infection, we demonstrate the critical role played by IL-12 in the rejection of male skin graft by female C57BL/6 syngeneic recipients and in the clearance of an immunogenic mastocytoma tumor variant by DBA/2 mice, but not in a parent to F1 immune aggression model nor in MOG-induced EAE, which was clearly prevented by anti-p40 mAb C17.8. Given this selective inhibition of IL-12, these mAbs provide new options for reassessing IL-12 function in vivo.     

Functional Characterization of Novel Mutations Affecting Survivin (BIRC5)‐Mediated Therapy Resistance in Head and Neck Cancer Patients

Survivin (BIRC5) is an acknowledged cancer therapy‐resistance factor and overexpressed in head and neck squamous cell carcinomas (HNSCC). Driven by its nuclear export signal (NES), Survivin shuttles between the nucleus and the cytoplasm, and is detectable in both cellular compartments in tumor biopsies. Although predominantly nuclear Survivin is considered a favorable prognostic disease marker for HNSCC patients, the underlying molecular mechanisms are not resolved. Hence, we performed immunohistochemical and mutational analyses using laser capture microdissection on HNSCC biopsies from patients displaying high levels of nuclear Survivin. We found somatic BIRC5 mutations, c.278T>C (p.Phe93Ser), c.292C>T (p.Leu98Phe), and c.288A>G (silent), in tumor cells, but not in corresponding normal tissues. Comprehensive functional characterization of the Survivin mutants by ectopic expression and microinjection experiments revealed that p.Phe93Ser, but not p.Leu98Phe inactivated Survivin's NES, resulted in a predominantly nuclear protein, and attenuated Survivin's dual cytoprotective activity against chemoradiation‐induced apoptosis. Notably, in xenotransplantation studies, HNSCC cells containing the p.Phe93Ser mutation responded significantly better to cisplatin‐based chemotherapy. Collectively, our results underline the disease relevance of Survivin's nucleocytoplasmic transport, and provide first evidence that genetic inactivation of Survivin's NES may account for predominantly nuclear Survivin and increased therapy response in cancer patients.     

IL-12p70、p40在子宫内膜异位症患者腹腔液中的表达及意义

目的　通过测定子宫内膜异位症患者腹腔液中IL - 12 p70及IL - 12p4 0的水平 ,探讨其与子宫内膜异位症发生发展的关系。方法　采用酶联免疫吸附法 (ELISA)测定 2 0例子宫内膜异位症患者 (内异症组 )及 2 0例卵巢良性肿瘤患者(对照组 )腹腔液中IL - 12p70、IL - 12 p4 0的水平。比较两组间两个细胞因子水平以探讨它们与内异症发生的关系 ,并探讨其是否与疾病的进展有关。结果　腹腔液中IL - 12 p70水平内异症组稍低于对照组 ,但两者之间差异无显著性 (P >0 0 5 ) ;IL- 12 p4 0水平内异症组明显高于对照组 ,差异有显著性 (P <0 0 5 ) ;内异症组中 (Ⅰ～Ⅱ期 )患者IL - 12 p4 0水平较 (Ⅲ～Ⅳ期 )患者稍高 ,但两者差异并无显著性 (P >0 0 5 ) ;而 (Ⅰ～Ⅱ期 )患者IL - 12 p70水平明显高于 (Ⅲ～Ⅳ期 )患者 ,差异有显著性 (P <0 0 5 )。结论　内异症患者腹腔液中IL - 12p4 0水平的增高可能是自然杀伤 (NK)细胞功能受损的部分原因 ,从而可能与子宫内膜异位症的发生有一定关系 ;腹腔液中IL - 12 p70水平随内异症疾病程度进展而下降 ,表明其可能是内异症中NK细胞功能进一步下降的原因之一 ,从而可能是促使内异症进一步发展的原因之一。     

Effects of Different Variants in the ENPP1 Gene on the Functional Properties of Ectonucleotide Pyrophosphatase/Phosphodiesterase Family Member 1

Ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (E‐NPP1), encoded by ENPP1, is a plasma membrane protein that generates inorganic pyrophosphate (PP_i), a physiologic inhibitor of hydroxyapatite formation. In humans, variants in ENPP1 are associated with generalized arterial calcification of infancy, an autosomal‐recessive condition causing premature onset of arterial calcification and intimal proliferation resulting in stenoses. ENPP1 variants also cause pseudoxanthoma elasticum characterized by ectopic calcification of soft connective tissues. To determine the functional impact of ENPP1 missense variants, we analyzed 13 putative pathogenic variants in vitro regarding their functional properties, that is, activity, localization, and PP_i generation. Transfection of eight of the 13 variants led to complete loss of NPP activity, whereas four mutants (c.1412A > G, p.Tyr471Cys; c.1510A > C, p.Ser504Arg; c.1976A > G, p.Tyr659Cys; c.2330A > G, p.His777Arg) showed residual activity compared with wild‐type E‐NPP1. One putative pathologic variant (c.2462 G > A, p.Arg821His) showed normal activity. The five mutants with normal or residual E‐NPP1 enzyme activity were still able to generate PP_i and localized in the plasma membrane. In this study, we identified a functional ENPP1 polymorphism, which was expected to be pathogenic till now. Furthermore, we identified four mutants (p.Tyr471Cys, p.Ser504Arg, p.Tyr659Cys, p.His777Arg) with residual E‐NPP1 function, which would be potential therapeutical targets for conformational‐stabilizing agents.     

Short stature and growth hormone deficiency in a subset of patients with Potocki–Lupski syndrome: Expanding the phenotype of PTLS

Potocki–Lupski Syndrome (PTLS, MIM 610883), or duplication of chromosome 17p11.2, is a clinically recognizable condition characterized by infantile hypotonia, failure to thrive, developmental delay, intellectual disability, and congenital anomalies. Short stature, classified as greater than two standard deviations below the mean, has not previously been considered a major feature of PTLS. Retrospective chart review on a cohort of 37 individuals with PTLS was performed to investigate the etiology of short stature. Relevant data included anthropometric measurements, insulin growth factor‐1 (IGF‐1), insulin‐like growth factor binding protein 3 (IGFBP‐3), growth hormone (GH) stimulation testing, blood glucose levels, brain MRI, and bone age. Approximately 25% (9/37) of individuals with PTLS had short stature. Growth hormone deficiency (GHD) was definitively identified in two individuals. These two PTLS patients with growth hormone deficiency, as well as three others with short stature and no documented GHD, received growth hormone and obtained improvement in linear growth. One individual was identified to have pituitary abnormalities on MRI and had complications of hypoglycemia due to unrecognized GHD. Individuals with PTLS can benefit from undergoing evaluation for GHD should they present with short stature or hypoglycemia. Early identification of GHD could facilitate potential therapeutic benefit for individuals with PTLS, including linear growth, musculoskeletal, and in cases of hypoglycemia, potentially cognitive development as well.