Familial multiple discoid fibromas is linked to a locus on chromosome 5 including the FNIP1 gene.

Previously, we reported a series of families presenting with trichodiscomas, inherited in an autosomal dominant pattern. The phenotype was named familial multiple discoid fibromas (FMDF). The genetic cause of FMDF remained unknown so far. Trichodiscomas are skin lesions previously reported to be part of the same spectrum as the fibrofolliculoma observed in Birt-Hogg-Dubé syndrome (BHD), an inherited disease caused by pathogenic variants in the FLCN gene. Given the clinical and histological differences with BHD and the exclusion of linkage with the FLCN locus, the phenotype was concluded to be distinct from BHD. We performed extensive clinical evaluations and genetic testing in ten families with FMDF. We identified a FNIP1 frameshift variant in nine families and genealogical studies showed common ancestry for eight families. Using whole exome sequencing, we identified six additional rare variants in the haplotype surrounding FNIP1, including a missense variant in the PDGFRB gene that was found to be present in all tested patients with FMDF. Genome-wide linkage analysis showed that the locus on chromosome 5 including FNIP1 was the only region reaching the maximal possible LOD score. We concluded that FMDF is linked to a haplotype on chromosome 5. Additional evaluations in families with FMDF are required to unravel the exact genetic cause underlying the phenotype. When evaluating patients with multiple trichodisomas without a pathogenic variant in the FLCN gene, further genetic testing is warranted and can include analysis of the haplotype on chromosome 5.

5q11.2 deletion syndrome revisited-Further narrowing of the smallest region of overlap for the main clinical characteristics of the syndrome.

Microdeletions at 5q11.2 are rare. Subjects show a phenotypic spectrum that overlaps CHARGE syndrome and 22q11.2 deletion syndrome. A growing number of subjects present with learning difficulty and/or intellectual disability, immune deficiency, congenital heart malformation, and dysmorphism. DHX29 and IL6ST have been proposed as candidate genes for the development of the major clinical manifestations. We present a new case and narrow down the shortest region of overlap to evaluate possible candidate genes. Our case does not present developmental delay or immune deficiency indicating a reduced penetrance for some of the main clinical manifestations. The shortest region of overlap between subjects with deletions at 5q11.2 is approximately 450 kb (position 54.3-54.7 Mb). The narrowed region comprises 10 protein coding genes, including DHX29. DHX29 is a strong candidate gene for the main features of 5q11.2-microdeletion syndrome; however, our findings suggest a joined impact of several genes as the cause of the syndrome.

Chondrodysplasia, enchondromas and a chest deformity causing severe pulmonary morbidity in a boy with a PTHLH duplication: A case report.

Parathyroid hormone-like hormone (PTHLH) plays an important role in bone formation. Several skeletal dysplasias have been described that are associated with disruption of PTHLH functioning. Here we report on a new patient with a 898 Kb duplication on chromosome 12p11.22 including the PTHLH gene. The boy has multiple skeletal abnormalities including chondrodysplasia, lesions radiographically resembling enchondromas and posterior rib deformities leading to a severe chest deformity. Severe pulmonary symptoms were thought to be caused by limited mobility and secondary sputum evacuation problems due to the chest deformity. Imaging studies during follow-up revealed progression of the number of skeletal lesions over time. This case extends the phenotypic spectrum associated with copy number variation of PTHLH.

CACNA1I gain-of-function mutations differentially affect channel gating and cause neurodevelopmental disorders.

T-type calcium channels (Cav3.1 to Cav3.3) regulate low-threshold calcium spikes, burst firing and rhythmic oscillations of neurons and are involved in sensory processing, sleep, and hormone and neurotransmitter release. Here, we examined four heterozygous missense variants in CACNA1I, encoding the Cav3.3 channel, in patients with variable neurodevelopmental phenotypes. The p.(Ile860Met) variant, affecting a residue in the putative channel gate at the cytoplasmic end of the IIS6 segment, was identified in three family members with variable cognitive impairment. The de novo p.(Ile860Asn) variant, changing the same amino acid residue, was detected in a patient with severe developmental delay and seizures. In two additional individuals with global developmental delay, hypotonia, and epilepsy, the variants p.(Ile1306Thr) and p.(Met1425Ile), substituting residues at the cytoplasmic ends of IIIS5 and IIIS6, respectively, were found. Because structure modelling indicated that the amino acid substitutions differentially affect the mobility of the channel gate, we analysed possible effects on Cav3.3 channel function using patch-clamp analysis in HEK293T cells. The mutations resulted in slowed kinetics of current activation, inactivation, and deactivation, and in hyperpolarizing shifts of the voltage-dependence of activation and inactivation, with Cav3.3-I860N showing the strongest and Cav3.3-I860M the weakest effect. Structure modelling suggests that by introducing stabilizing hydrogen bonds the mutations slow the kinetics of the channel gate and cause the gain-of-function effect in Cav3.3 channels. The gating defects left-shifted and increased the window currents, resulting in increased calcium influx during repetitive action potentials and even at resting membrane potentials. Thus, calcium toxicity in neurons expressing the Cav3.3 variants is one likely cause of the neurodevelopmental phenotype. Computer modelling of thalamic reticular nuclei neurons indicated that the altered gating properties of the Cav3.3 disease variants lower the threshold and increase the duration and frequency of action potential firing. Expressing the Cav3.3-I860N/M mutants in mouse chromaffin cells shifted the mode of firing from low-threshold spikes and rebound burst firing with wild-type Cav3.3 to slow oscillations with Cav3.3-I860N and an intermediate firing mode with Cav3.3-I860M, respectively. Such neuronal hyper-excitability could explain seizures in the patient with the p.(Ile860Asn) mutation. Thus, our study implicates CACNA1I gain-of-function mutations in neurodevelopmental disorders, with a phenotypic spectrum ranging from borderline intellectual functioning to a severe neurodevelopmental disorder with epilepsy.

upd(20)mat is a rare cause of the Silver-Russell-syndrome-like phenotype: Two unrelated cases and screening of large cohorts.

Silver-Russell syndrome (SRS) is an imprinting disorder characterized by prenatal and postnatal growth retardation, relative macrocephaly, feeding difficulties and body asymmetry. Recently, upd(20)mat has been identified in few patients with SRS-like features, suggestive of a new imprinting disorder characterized by prenatal and postnatal growth failure. Here, we describe two male patients with upd(20) and feeding difficulties, prenatal and postnatal growth retardation and normal cognitive development. During pregnancy, confined placental mosaicism for trisomy 20 was detected in one of the patients but was not investigated further until identification of upd(20)mat in the neonatal period. To evaluate whether upd(20)mat should be part of the first trier genetic diagnostic in patients with growth retardation, we screened a large cohort of patients (n = 673) referred to our laboratories for SRS-testing without detecting any upd(20). Our results, along with the existing evidence, indicate that upd(20)mat is a very rare cause of growth retardation, but should be followed up when confined placental mosaicism for trisomy 20 mosaicism is observed during pregnancy.

Loss-of-function variants in myocardin cause congenital megabladder in humans and mice.

Myocardin (MYOCD) is the founding member of a class of transcriptional coactivators that bind the serum-response factor to activate gene expression programs critical in smooth muscle (SM) and cardiac muscle development. Insights into the molecular functions of MYOCD have been obtained from cell culture studies, and to date, knowledge about in vivo roles of MYOCD comes exclusively from experimental animals. Here, we defined an often lethal congenital human disease associated with inheritance of pathogenic MYOCD variants. This disease manifested as a massively dilated urinary bladder, or megabladder, with disrupted SM in its wall. We provided evidence that monoallelic loss-of-function variants in MYOCD caused congenital megabladder in males only, whereas biallelic variants were associated with disease in both sexes, with a phenotype additionally involving the cardiovascular system. These results were supported by cosegregation of MYOCD variants with the phenotype in 4 unrelated families by in vitro transactivation studies in which pathogenic variants resulted in abrogated SM gene expression and by the finding of megabladder in 2 distinct mouse models with reduced Myocd activity. In conclusion, we have demonstrated that variants in MYOCD result in human disease, and the collective findings highlight a vital role for MYOCD in mammalian organogenesis.

Capecitabine-based treatment of a patient with a novel DPYD genotype and complete dihydropyrimidine dehydrogenase deficiency.

Fluoropyrimidines are frequently used anti-cancer drugs. It is known that patients with reduced activity of dihydropyrimidine dehydrogenase (DPD), the key metabolic enzyme in fluoropyrimidine inactivation, are at increased risk of developing severe fluoropyrimidine-related toxicity. Upfront screening for DPD deficiency and dose reduction in patients with partial DPD deficiency is recommended and improves patient safety. For patients with complete DPD deficiency, fluoropyrimidine-treatment has generally been discouraged. During routine pretreatment screening, we identified a 59-year-old patient with a sigmoid adenocarcinoma who proved to have a complete DPD deficiency. Genetic analyses showed that this complete absence of DPD activity was likely to be caused by a novel DPYD genotype, consisting of a combination of amplification of exons 17 and 18 of DPYD and heterozygosity for DPYD*2A. Despite absence of DPD activity, the patient was treated with capecitabine-based chemotherapy, but capecitabine dose was drastically reduced to 150 mg once every 5 days (0.8% of original dose). Pharmacokinetic analyses showed that the area under the concentration-time curve (AUC) and half-life of 5-fluorouracil were respectively tenfold and fourfold higher than control values of patients receiving capecitabine 850 mg/m2 . When extrapolating from the dosing schedule of once every 5 days to twice daily, the AUC of 5-fluorouracil was comparable to controls. Treatment was tolerated well for eight cycles by the patient without occurrence of capecitabine-related toxicity. This case report demonstrates that a more comprehensive genotyping and phenotyping approach, combined with pharmacokinetically-guided dose administration, enables save fluoropyrimidine-treatment with adequate drug exposure in completely DPD deficient patients.

Cytomegalovirus and human herpesvirus 8 DNA detection in peripheral blood monocytic cells of AIDS patients: correlations with the presence of Kaposi's sarcoma and CMV disease.

To establish the effect of the presence in blood cells of cytomegalovirus (CMV) and human herpesvirus 8 (HHV8) DNA, two herpesviruses that are activated frequently in AIDS patients, were selected from the Amsterdam Cohort Studies on HIV/AIDS 181 PBMC samples from patients with and without Kaposi's sarcoma (KS), and with and without CMV-related disease. The viral loads of both HHV8 and CMV were determined by real-time PCR at the time of diagnosis of AIDS. There was no significant difference in prevalence and load for CMV between the KS and non-KS patients. The variable related most strongly to KS was the presence of HHV8 DNA in PBMCs, whilst CMV DNA was related to the development of CMV disease and shortened survival. The frequency of detection of HHV8 increased when the patient presented with more severe KS symptoms at diagnosis, but detection of HHV8 DNA did not influence survival. Therefore, HHV8 and CMV DNA measured in the blood of AIDS patients, are each related mainly to the associated disease, and have no additional predictive value in these patients.

Dynamics of cytomegalovirus (CMV)-specific T cells in HIV-1-infected individuals progressing to AIDS with CMV end-organ disease.

BACKGROUND: Since cytomegalovirus (CMV) infection can cause serious clinical complications in immunocompromised individuals, we assessed cellular immune requirements for protection against CMV end-organ disease (CMV-EOD) in human immunodeficiency virus type 1 (HIV-1) infection. METHODS: Longitudinal samples from HIV-1-infected patients in the Amsterdam cohort were analyzed. Dynamics of CMV-specific CD8(+) and CD4(+) T cell responses were analyzed by 4-color fluorescence analysis using major histocompatibility class I CMV peptide-tetramers and by intracellular staining for perforin, granzyme B, and interferon (IFN)- gamma after stimulation with CMV-specific stimuli. CMV load was measured in parallel. RESULTS: In individuals progressing to acquired immunodeficiency syndrome with CMV-EOD, CMV-specific IFN- gamma -producing CD4(+) T cells disappeared during the year before onset of CMV-EOD. This disappearance was accompanied by a sharp increase in CMV load before onset of disease. Despite increasing CMV-specific CD8(+) T cell counts, decreasing CMV-specific IFN- gamma -producing CD8(+) T cell counts were found over time. In contrast, the percentage of CMV-specific perforin- and granzyme B-expressing CD8(+) T cells increased. CONCLUSIONS: Our data indicate that insufficient help of CD4(+) T cells may cause loss of IFN- gamma -producing CD8(+) T cells and loss of control of CMV dissemination. Increasing CMV-infected cell counts in the face of high CMV-specific perforin- and granzyme B-expressing CD8(+) T cell counts may explain the immune pathological characteristics of CMV disease.

Retrospective, longitudinal analysis of serum human herpesvirus-8 viral DNA load in AIDS-related Kaposi's sarcoma patients before and after diagnosis.

Human herpesvirus 8 (HHV-8) is detected more often in patients progressing towards Kaposi's sarcoma (KS) than in patients who do not develop the disease, suggesting that the level of viremia might be associated with Kaposi's sarcoma disease and progression. Longitudinal serum samples from 19 AIDS-Kaposi's sarcoma patients, ranging from 2 years before KS till 2 years after KS diagnosis were tested. No correlation was found between viral load and progression to KS, or disease stage.

An IL-8 gene promoter polymorphism is associated with the risk of the development of AIDS-related Kaposi's sarcoma: a case-control study.

In a case-control study, we studied the effect of a single nucleotide polymorphism in the IL-8 promoter on the risk of the development of AIDS-related Kaposi's sarcoma (KS). KS developed in 46% of individuals with the TT genotype and in 66% of AA/AT genotypes (P=0.038). Patients with TT genotype were rarely affected with visceral KS (7% versus 36%; P=0.06), which suggests that carriers of the TT genotype are protected from (severe) KS development.

Human herpesvirus 8 load in matched serum and plasma samples of patients with AIDS-associated Kaposi's sarcoma.

Human herpesvirus 8 (HHV-8) (or Kaposi's sarcoma [KS]-associated herpesvirus) is associated with all forms of KS. HHV-8 DNA load in peripheral blood mononuclear cells (PBMCs) of KS patients has been shown to correlate with the clinical stage of the disease. Studies have been done to assess the HHV-8 viral load in different sample types from KS patients and its clinical relevance. This paper describes the design and evaluation of a quantitative real-time (TaqMan) PCR assay for routine diagnosis of HHV-8 infection. The linear dynamic range was 5 to 5 x 10(6) copies of HHV-8 DNA (r(2) > 0.99). The assay is very sensitive, specific, and easily reproducible (less than 2% variability) and can be used for different clinical samples, such as serum, plasma, and PBMCs. The question of which clinical sample, serum or plasma, is preferable for HHV8 DNA testing was addressed, using this newly developed real-time PCR assay. From 85 patients with diagnosed AIDS-KS, matched plasma and serum samples were collected. Of the 85 patients tested, 35 were positive for HHV-8 DNA in both plasma and serum (41%), 8 were positive in serum but not plasma, and 7 had detectable HHV-8 DNA only in plasma. The HHV-8 load was similar in both plasma and serum, and no significant difference was found. However, more inhibition was seen in the plasma samples with the use of a system quality control, seal herpesvirus type 1. Therefore, our results suggest that serum is the preferred material for HHV-8 load testing, since there is less possible hindrance in the amplification than with plasma.

Latent and lytic HHV-8 mRNA expression in PBMCs and Kaposi's sarcoma skin biopsies of AIDS Kaposi's sarcoma patients.

Human herpes virus 8 (HHV-8) is associated with all clinical forms of Kaposi's sarcoma. HHV-8 DNA is present in Kaposi's sarcoma biopsies and is observed regularly in saliva and less consistently in blood of Kaposi's sarcoma patients. The expression pattern of latent (ORF 73) and lytic (vGCR, vBcl-2, and vIL-6) HHV-8 mRNA was studied in peripheral blood mononuclear cell (PBMC) samples and Kaposi's sarcoma skin biopsies from 11 AIDS Kaposi's sarcoma patients with four different nucleic acid sequence-based amplification (NASBA) assays. Patients were divided into groups according to the clinical stage of Kaposi's sarcoma (stage I-IV). All biopsies were positive for two or more of the mRNA measured. No clear difference could be seen in the expression pattern in the lesions of the different clinical stages. In the corresponding PBMC samples, very little or no mRNA was measurable in the patients with Kaposi's sarcoma stage I or II, whereas patients with more advanced Kaposi's sarcoma (stage III or IV) had more detectable mRNA in the PBMCs. Thus, the HHV-8 DNA load in the PBMCs increases in more advanced Kaposi's sarcoma, as does the frequency of mRNA detection in PBMCs.

Development of real-time NASBA assays with molecular beacon detection to quantify mRNA coding for HHV-8 lytic and latent genes.

BACKGROUND: Human herpesvirus-8 (HHV-8) is linked to the pathogenesis of Kaposi's sarcoma (KS), and the HHV-8 DNA load in peripheral blood mononuclear cells (PBMC) is associated with the clinical stage of KS. To examine the expression of HHV-8 in PBMC, four HHV-8 mRNA specific NASBA assays were developed METHODS: We have developed four quantitative nucleic acid sequence-based amplification assays (NASBA-QT) specifically to detect mRNA coding for ORF 73 (latency-associated nuclear antigen, LANA), vGCR (a membrane receptor), vBcl-2 (a viral inhibitor of apoptosis) and vIL-6 (a viral growth factor). The NASBA technique amplifies nucleic acids without thermocycling and mRNA can be amplified in a dsDNA background. A molecular beacon is used during amplification to enable real-time detection of the product. The assays were tested on PBMC samples of two AIDS-KS patients from the Amsterdam Cohort. RESULTS: For all four assays, the limit of detection (LOD) of 50 molecules and the limit of quantification (LOQ) of 100 molecules were determined using in vitro transcribed RNA. The linear dynamic range was 50 to 10(7) molecules of HHV-8 mRNA. We found HHV-8 mRNA expression in 9 out of the 10 tested samples. CONCLUSION: These real-time NASBA assays with beacon detection provide tools for further study of HHV-8 expression in patient material.