Identification and Molecular Characterization of a Novel Large-Scale Variant (Exons 4_18 Loss) in the LDLR Gene as a Cause of Familial Hypercholesterolaemia in an Italian Family.

Next-generation sequencing (NGS) is nowadays commonly used for clinical purposes, and represents an efficient approach for the molecular diagnosis of familial hypercholesterolemia (FH). Although the dominant form of the disease is mostly due to the low-density lipoprotein receptor (LDLR) small-scale pathogenic variants, the copy number variations (CNVs) represent the underlying molecular defects in approximately 10% of FH cases. Here, we reported a novel large deletion in the LDLR gene involving exons 4-18, identified by the bioinformatic analysis of NGS data in an Italian family. A long PCR strategy was employed for the breakpoint region analysis where an insertion of six nucleotides (TTCACT) was found. Two Alu sequences, identified within intron 3 and exon 18, could underlie the identified rearrangement by a nonallelic homologous recombination (NAHR) mechanism. NGS proved to be an effective tool suitable for the identification of CNVs, together with small-scale alterations in the FH-related genes. For this purpose, the use and implementation of this cost-effective, efficient molecular approach meets the clinical need for personalized diagnosis in FH cases.

Lysosomal lipase deficiency: molecular characterization of eleven patients with Wolman or cholesteryl ester storage disease.

Wolman Disease (WD) and cholesteryl ester storage disease (CESD) represent two distinct phenotypes of the same recessive disorder caused by the complete or partial deficiency of lysosomal acidic lipase (LAL), respectively. LAL, encoded by the LIPA gene, hydrolyzes cholesteryl esters derived from cell internalization of plasma lipoproteins. WD is a rapidly progressive and lethal disease characterized by intestinal malabsorption, hepatic and adrenal failure. CESD is characterized by hepatic fibrosis, hyperlipidemia and accelerated atherosclerosis. Aim of the study was the identification of LIPA mutations in three WD and eight CESD patients. The WD patients, all deceased before the first year of age, were homozygous for two novel mutations (c.299+1G>A and c.419G>A) or a mutation (c.796G>T) previously reported as compound heterozygosity in a CESD patient. The two mutations (c.419G>A and c.796G>T) resulting in truncated proteins (p.W140* and p.G266*) and the splicing mutation (c.229+1G>A) were associated with undetectable levels of LIPA mRNA in fibroblasts. All eight CESD patients carried the common mutation c.894G>A known to result not only in a major non-functional transcript with the skipping of exon 8 (p.S275_Q298del), but also in a minor normally spliced transcript producing 5-10% residual LAL activity. The c.894G>A mutation was found in homozygosity in four patients and, as compound heterozygosity, in association with a known (p.H295Y and p.G342R) or a novel (p.W140*) mutation in four other CESD patients. Segregation analysis performed in all patients harboring c.895G>A showed its occurrence on the same haplotype suggesting a common founder ancestor. The other WD and CESD mutations were associated with different haplotypes.

Novel mutations of ABCA1 transporter in patients with Tangier disease and familial HDL deficiency.

The objective of the study was the characterization of ABCA1 gene mutations in 10 patients with extremely low HDL-cholesterol. Five patients (aged 6 months to 76 years) presented with splenomegaly and thrombocytopenia suggesting the diagnosis of Tangier disease (TD). Three of them were homozygous for novel mutations either in intron (c.4465-34A>G) or in exons (c.4376delT and c.5449C>T), predicted to encode truncated proteins. One patient was compound heterozygous for a nucleotide insertion (c.1758_1759insG), resulting in a truncated protein and for a nucleotide substitution c.4799A>G, resulting in a missense mutation (p.H1600R). The last TD patient, found to be heterozygous for a known mutation (p.D1009Y), had a complete defect in ABCA1-mediated cholesterol efflux in fibroblasts, suggesting the presence of a second undetected mutant allele. Among the other patients, four were asymptomatic, but one, with multiple risk factors, had severe peripheral artery disease. Three of these patients were heterozygous for known mutations (p.R130K+p.N1800H, p.R1068C, p.N1800H), while two were carriers of novel mutations (c.1195-27G>A and c.396_397insA), predicted to encode truncated proteins. The pathogenic effect of the two intronic mutations (c. 1195-27G>A and c.4465-34A>G) was demonstrated by the analysis of the transcripts of splicing reporter mutant minigenes expressed in COS-1 cells. Both mutations activated an intronic acceptor splice site which resulted in a partial intron retention in mature mRNA with the production of truncated proteins. This study confirms the allelic heterogeneity of TD and suggests that the diagnosis of TD must be considered in patients with an unexplained splenomegaly, associated with thrombocytopenia and hypocholesterolemia.

HDL-mediated reduction of cholesterol content inhibits the proliferation of prostate cancer cells induced by LDL: Role of ABCA1 and proteasome inhibition.

High-density lipoproteins (HDL) are well known for their atheroprotective function, mainly due to their ability to remove cell cholesterol and to exert antioxidant and anti-inflammatory activities. Through the same mechanisms HDL could also affect the development and progression of tumors. Cancer cells need cholesterol to proliferate, especially in hormone-dependent tumors, as prostate cancer (PCa). Aim of the study was to investigate the ability of HDL to modulate cholesterol content and metabolism in androgen receptor (AR)-positive and AR-null PCa cell lines and the consequences on cell proliferation. HDL inhibited colony formation of LNCaP and PC3 cells. HDL reduced cell cholesterol content and proliferation of LNCaP cells loaded with low-density lipoproteins but were not effective on PC3 cells. Here, the expression of the ATP-binding cassette transporter A1 (ABCA1) was markedly reduced due to proteasome degradation. Bortezomib, a proteasome inhibitor, restored ABCA1 expression and HDL ability to promote cholesterol removal from PC3; consequently, HDL inhibited the proliferation of PC3 cells induced by LDL only after bortezomib pre-treatment. In conclusion, the antiproliferative activity of HDL on AR-positive and AR-null PCa cells also rely on cholesterol removal, a process in which the ABCA1 transporter plays a key role.

Comparison of two polygenic risk scores to identify non-monogenic primary hypocholesterolemias in a large cohort of Italian hypocholesterolemic subjects.

BACKGROUND: Primary Hypobetalipoproteinemias (HBL) are a group of dominant and recessive monogenic genetic disorders caused by mutations in APOB, PCSK9, ANGPTL3, MTTP, Sar1b genes and characterized by plasma levels of total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C) and apolipoprotein B (apoB) below the 5th percentile of the distribution in a given population. Mutations in the candidate genes account only for a small proportion of subjects with HBL suggesting a role for a polygenic contribution to the low cholesterol phenotype. OBJECTIVE: To explore the complex genetic architecture of HBL we compared two polygenic risk scores in order to assess the role of the polygenic burden and the differences in the clinical phenotype between monogenic and polygenic HBL; we studied a cohort of 170 subjects with primary HBL referred over a 25-year period to 2 Italian reference centers have been studied. METHODS: The genetic analyses have been based on: Sanger sequencing, in-house NGS customized panel and two scores, PRS1 and PRS2 for the polygenic burden. RESULTS: Sixty 60 (35%) and 63 (37%) subjects had a monogenic and polygenic HBL respectively. LDL-C plasma levels were significantly lower in monogenic HBL (30.87 ± 3.12 mg/dl) compared with the non-monogenic HBL (42.80 ± 2.18 mg/dl) (p<0.002) with no differences in the percentage of fatty liver. CONCLUSION: Only PRS1 is effective in detecting polygenic HBL while PRS2 does not improve the polygenic diagnosis.

Characterization of New ATM Deletion Associated with Hereditary Breast Cancer.

Next-generation sequencing (NGS)-based cancer risk screening with multigene panels has become the most successful method for programming cancer prevention strategies. ATM germ-line heterozygosity has been described to increase tumor susceptibility. In particular, families carrying heterozygous germ-line variants of ATM gene have a 5- to 9-fold risk of developing breast cancer. Recent studies identified ATM as the second most mutated gene after CHEK2 in BRCA-negative patients. Nowadays, more than 170 missense variants and several truncating mutations have been identified in ATM gene. Here, we present the molecular characterization of a new ATM deletion, identified thanks to the CNV algorithm implemented in the NGS analysis pipeline. An automated workflow implementing the SOPHiA Genetics' Hereditary Cancer Solution (HCS) protocol was used to generate NGS libraries that were sequenced on Illumina MiSeq Platform. NGS data analysis allowed us to identify a new inactivating deletion of exons 19-27 of ATM gene. The deletion was characterized both at the DNA and RNA level.

Circulating mucosal-associated invariant T cells identify patients responding to anti-PD-1 therapy.

Immune checkpoint inhibitors are used for treating patients with metastatic melanoma. Since the response to treatment is variable, biomarkers are urgently needed to identify patients who may benefit from such therapy. Here, we combine single-cell RNA-sequencing and multiparameter flow cytometry to assess changes in circulating CD8+ T cells in 28 patients with metastatic melanoma starting anti-PD-1 therapy, followed for 6 months: 17 responded to therapy, whilst 11 did not. Proportions of activated and proliferating CD8+ T cells and of mucosal-associated invariant T (MAIT) cells are significantly higher in responders, prior to and throughout therapy duration. MAIT cells from responders express higher level of CXCR4 and produce more granzyme B. In silico analysis support MAIT presence in the tumor microenvironment. Finally, patients with >1.7% of MAIT among peripheral CD8+ population show a better response to treatment. Our results thus suggest that MAIT cells may be considered a biomarker for patients responding to anti-PD-1 therapy.

Functional analysis of two novel splice site mutations of APOB gene in familial hypobetalipoproteinemia.

Familial hypobetalipoproteinemia (FHBL) is a co-dominant disorder characterized by reduced plasma levels of low density lipoprotein cholesterol (LDL-C) and its protein constituent apolipoprotein B (apoB), which may be due to mutations in APOB gene, mostly located in the coding region of this gene. We report two novel APOB gene mutations involving the acceptor splice site of intron 11 (c.1471-1G>A) and of intron 23 (c.3697-1G>C), respectively, which were identified in two patients with heterozygous FHBL associated with severe fatty liver disease. The effects of these mutations on APOB pre-mRNA splicing were assessed in COS-1 cells expressing the mutant APOB minigenes. The c.1471-1G>A APOB minigene generated two abnormal mRNAs. In one mRNA the entire intron 11 was retained; in the other mRNA exon 11 joined to exon 12, in which the first nucleotide was deleted due to the activation of a novel acceptor splice site. The predicted products of these mRNAs are truncated proteins of 546 and 474 amino acids, designated apoB-12.03 and apoB-10.45, respectively. The c.3697-1G>C APOB minigene generated a single abnormal mRNA in which exon 23 joined to exon 25, with the complete skipping of exon 24. This abnormal mRNA is predicted to encode a truncated protein of 1220 amino acids, designated apoB-26.89. These splice site mutations cause the formation of short truncated apoBs, which are not secreted into the plasma as lipoprotein constituents. This secretion defect is the major cause of severe fatty liver observed in carriers of these mutations.

Novel mutations of SAR1B gene in four children with chylomicron retention disease.

BACKGROUND: Intestinal lipid malabsorption, resulting from an impaired formation or secretion of chylomicrons and associated with severe hypobetalipoproteinemia (HBL), may be due to biallelic mutations in APOB (homozygous FHBL type-1), MTTP (abetalipoproteinemia), or SAR1B (chylomicron retention disease). OBJECTIVE: We investigated four children, each born from consanguineous parents, presenting with steatorrhea, malnutrition, accumulation of lipids in enterocytes, and severe hypocholesterolemia with an apparent recessive transmission. METHODS: We sequenced a panel of genes whose variants may be associated with HBL. RESULTS: Case 1, a 9-month-old male, was found to be homozygous for a SAR1B variant (c.49 C>T), predicted to encode a truncated Sar1b protein devoid of function (p.Gln17*). Case 2, a 4-year-old male, was found to be homozygous for a SAR1B missense variant [c.409 G>C, p.(Asp137His)], which affects a highly conserved residue close to the Sar1b guanosine recognition site. Case 3, a 6-year-old male, was found to be homozygous for an ∼6 kb deletion of the SAR1B gene, which eliminates exon 2; this deletion causes the loss of the ATG translation initiation codon in the SAR1B mRNA. The same homozygous mutation was found in an 11-month-old child (case 4) who was related to case 3. CONCLUSIONS: We report 4 children with intestinal lipid malabsorption were found to have chylomicron retention disease due to 3 novel variants in the SAR1B gene.

In vitro functional characterization of splicing variants of the APOB gene found in familial hypobetalipoproteinemia.

BACKGROUND: Familial hypobetalipoproteinemia type 1 (FHBL-1) is a codominant disorder characterized by greatly reduced plasma levels of total cholesterol, low-density lipoprotein cholesterol, and apolipoprotein B. Rare exonic pathogenic variants of APOB gene (nonsense variants, minute deletions/insertions and nonsynonymous variants) have been frequently reported in subjects with FHBL-1. Also, rare intronic variants of APOB located at intron/exon junctions and assumed to affect splicing have been reported. However, the pathogenicity of most of these intronic variants remains to be established. OBJECTIVE: The objective of this study was the in vitro functional characterization of six splicing variants of APOB gene identified in seven putative FHBL-1 heterozygotes. METHODS: ApoB minigenes harboring each variant were expressed in COS-1 cells and their transcripts were sequenced. RESULTS: Four novel variants (c.237+1G>A, c.818+5G>A, c.3000-1G>T, and c.3842+1G>A), predicted in silico to obliterate splice site activity, were found to generate abnormal transcripts. The abnormal transcripts were generated by the activation of cryptic splice sites or exon skipping. All these transcripts harbored a premature termination codon and were predicted to encode truncated apoBs devoid of function. The predicted translation products were: i) p.(Lys41Serfs*2) and p.(Val80Ilefs*10) for c.237+1G>A; ii) p.(Asn274*) for c.818+5G>A; iii) p.(Leu1001Alafs*10) for c.3000-1G>T, and iv) p.(Ser1281Argfs*2) for c.3842+1G>A. Two previously annotated rare variants (c.905-15C>G and c.1618-4G>A) with uncertain effect in silico were found to generate only wild-type transcripts. CONCLUSIONS: These in vitro minigene expression studies support the assignment of pathogenicity to four novel splice site variants of APOB gene found in FHBL-1.

The study of familial hypercholesterolemia in Italy: A narrative review.

In this review we outline our experience in the clinical and molecular diagnosis of familial hypercholesterolemia (FH), built up over more than three decades. We started our work by selecting FH patients on the basis of stringent clinical criteria, including extensive family studies. In most patients we confirmed the clinical diagnosis by showing a reduced LDLR activity in skin fibroblasts. After the isolation of LDLR cDNA and the characterization of the corresponding gene by the Dallas group, we started a systematic molecular investigation of our patients first using Southern blotting, and, subsequently Sanger sequencing. Up to now we have been able to identify 260 mutations of LDLR gene in more than 1000 genotyped FH patients, including 68 homozygotes. During this survey we identified 13 mutation clusters located in different geographical districts, which gave us the chance to compare the phenotype of patients carrying the most common mutations. We also found that mutations in APOB and PCSK9 genes were a rare cause of FH in our cohort. Despite our efforts, we failed to identify mutations in candidate genes in ∼20% of cases of definite FH. An exome-wide study, conducted within the context of an international collaboration, excluded the presence of other major genes in our unexplained FH cases. Recently, we have adopted sequencing technology of the next generation (NGS) with the parallel sequencing of a panel of FH targeted genes as a way of obtaining a more comprehensive picture of the gene variants potentially involved in the disease.

Timely diagnosis of sitosterolemia by next generation sequencing in two children with severe hypercholesterolemia.

BACKGROUND AND AIMS: Severe hypercholesterolemia associated or not with xanthomas in a child may suggest the diagnosis of homozygous autosomal dominant hypercholesterolemia (ADH), autosomal recessive hypercholesterolemia (ARH) or sitosterolemia, depending on the transmission of hypercholesterolemia in the patient's family. Sitosterolemia is a recessive disorder characterized by high plasma levels of cholesterol and plant sterols due to mutations in the ABCG5 or the ABCG8 gene, leading to a loss of function of the ATP-binding cassette (ABC) heterodimer transporter G5-G8. METHODS: We aimed to perform the molecular characterization of two children with severe primary hypercholesterolemia. RESULTS: Case #1 was a 2 year-old girl with high LDL-cholesterol (690 mg/dl) and tuberous and intertriginous xanthomas. Case #2 was a 7 year-old boy with elevated LDL-C (432 mg/dl) but no xanthomas. In both cases, at least one parent had elevated LDL-cholesterol levels. For the molecular diagnosis, we applied targeted next generation sequencing (NGS), which unexpectedly revealed that both patients were compound heterozygous for nonsense mutations: Case #1 in ABCG5 gene [p.(Gln251*)/p.(Arg446*)] and Case #2 in ABCG8 gene [p.(Ser107*)/p.(Trp361*)]. Both children had extremely high serum sitosterol and campesterol levels, thus confirming the diagnosis of sisterolemia. A low-fat/low-sterol diet was promptly adopted with and without the addition of ezetimibe for Case #1 and Case #2, respectively. In both patients, serum total and LDL-cholesterol decreased dramatically in two months and progressively normalized. CONCLUSIONS: Targeted NGS allows the rapid diagnosis of sitosterolemia in children with severe hypercholesterolemia, even though their family history does not unequivocally suggest a recessive transmission of hypercholesterolemia. A timely diagnosis is crucial to avoid delays in treatment.

Incidental finding of severe hypertriglyceridemia in children. Role of multiple rare variants in genes affecting plasma triglyceride.

BACKGROUND: The incidental finding of severe hypertriglyceridemia (HyperTG) in a child may suggest the diagnosis of familial chylomicronemia syndrome (FCS), a recessive disorder of the intravascular hydrolysis of triglyceride (TG)-rich lipoproteins. FCS may be due to pathogenic variants in lipoprotein lipase (LPL), as well as in other proteins, such as apolipoprotein C-II and apolipoprotein A-V (activators of LPL), GPIHBP1 (the molecular platform required for LPL activity on endothelial surface) and LMF1 (a factor required for intracellular formation of active LPL). OBJECTIVE: Molecular characterization of 5 subjects in whom HyperTG was an incidental finding during infancy/childhood. METHODS: We performed the parallel sequencing of 20 plasma TG-related genes. RESULTS: Three children with severe HyperTG were found to be compound heterozygous for rare pathogenic LPL variants (2 nonsense, 3 missense, and 1 splicing variant). Another child was found to be homozygous for a nonsense variant of APOA5, which was also found in homozygous state in his father with longstanding HyperTG. The fifth patient with a less severe HyperTG was found to be heterozygous for a frameshift variant in LIPC resulting in a truncated Hepatic Lipase. In addition, 1 of the patients with LPL deficiency and the patient with APOA-V deficiency were also heterozygous carriers of a pathogenic variant in LIPC and LPL gene, respectively, whereas the patient with LIPC variant was also a carrier of a rare APOB missense variant. CONCLUSIONS: Targeted parallel sequencing of TG-related genes is recommended to define the molecular defect in children presenting with an incidental finding of HyperTG.

Clinical and genetic features of 3 patients with familial chylomicronemia due to mutations in GPIHBP1 gene.

BACKGROUND: Familial chylomicronemia is a recessive disorder that may be due to mutations in lipoprotein lipase (LPL) and in other proteins such as apolipoprotein C-II and apolipoprotein A-V (activators of LPL), GPIHBP1 (the molecular platform required for LPL activity on endothelial surface), and LMF1 (a factor required for intracellular formation of active LPL). METHODS: We sequenced the familial chylomicronemia candidate genes in 2 adult females presenting long-standing hypertriglyceridemia and a history of acute pancreatitis. RESULTS: Both probands had plasma triglyceride >10 mmol/L but no mutations in the LPL gene. The sequence of the other candidate genes showed that one patient was homozygous for a novel missense mutation p.(Cys83Arg), and the other was homozygous for a previously reported nonsense mutation p.(Cys 89*), respectively, in GPIHBP1. Family screening showed that the hypertriglyceridemic brother of the p.(Cys83Arg) homozygote was also homozygous for this mutation. He had no history of pancreatitis. The p.(Cys83Arg) heterozygous carriers had normal triglyceride levels. The substitution of a cysteine residue in the Ly6 domain of GPIHBP1 is predicted to abolish one of the disulfide bridges required to maintain the structure of GPIHBP1. The p.(Cys89*) mutation results in a truncated protein devoid of function. CONCLUSIONS: Both mutant GPIHBP1 proteins are expected to be incapable of transferring LPL from the subendothelial space to the endothelial surface.

Phenotypic variability in 4 homozygous familial hypercholesterolemia siblings compound heterozygous for LDLR mutations.

BACKGROUND: Homozygous familial hypercholesterolemia is a rare clinical phenotype with a variable expression, which is characterized by extremely elevated plasma low-density lipoprotein (LDL), tendon and skin xanthomas, and a progressive atherosclerosis. In 95% of patients, homozygous familial hypercholesterolemia is due to mutations in low-density lipoprotein receptor (LDLR) gene, which abolish (receptor-negative) or greatly reduce (receptor-defective) LDLR function. OBJECTIVE: The objective of the study was the molecular and phenotypic characterization of 4 siblings with severe hypercholesterolemia. METHODS: The major LDL-related genes (LDLR, APOB, PCSK9, ANGPTL3, APOE, and APOC3) were sequenced. LDLR messenger RNA, isolated from leukocytes, was reverse transcribed and sequenced. RESULTS: The index cases were 24-year-old identical twin sisters with long-standing tendon xanthomas and high low-density lipoprotein cholesterol (LDL-C ∼10 mmol/L) but no coronary heart disease. They were carriers of 2 LDLR mutations: (1) a previously reported mutation [p.(G335S)] inherited from the mother who had LDL-C level within normal range; (2) a novel 24 bp deletion in exon 8/intron 8 junction inherited from the hypercholesterolemic (LDL-C 6.1 mmol/L) father. The deletion allele encodes an messenger RNA with a partial deletion of exon 8, whose translation product has an in-frame deletion of 17 amino acids [p.(Glu380_Gly396del)]. Family screening revealed that the 2 siblings of the twin sisters were also compound heterozygotes but had much lower LDL-C levels (8.2 and 7.1 mmol/L). The sequence of potential modifying genes showed that the 2 siblings and the mother of the twin sisters were heterozygous for a rare missense variant of apoB [p.(S2429T)], which might have an LDL-lowering effect. CONCLUSIONS: We report a rare event of 4 siblings found to be compound heterozygotes for 2 LDLR gene mutations but showing a different phenotype severity. The less severely affected siblings were carriers of a rare apoB missense variant.

Novel mutations in the GPIHBP1 gene identified in 2 patients with recurrent acute pancreatitis.

BACKGROUND: Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) has been demonstrated to be essential for the in vivo function of lipoprotein lipase (LPL), the major triglyceride (TG)-hydrolyzing enzyme involved in the intravascular lipolysis of TG-rich lipoproteins. Recently, loss-of-function mutations of GPIHBP1 have been reported as the cause of type I hyperlipoproteinemia in several patients. METHODS: Two unrelated patients were referred to our Lipid Units because of a severe hypertriglyceridemia and recurrent pancreatitis. We measured LPL activity in postheparin plasma and serum ApoCII and sequenced LPL, APOC2, and GPIHBP1. RESULTS: The 2 patients exhibited very low LPL activity not associated with mutations in LPL gene or with ApoCII deficiency. The sequence of GPIHBP1 revealed 2 novel point mutations. One patient (proband 1) was found to be homozygous for a C>A transversion in exon 3 resulting in the conversion of threonine to lysine at position 80 (p.Thr80Lys). The other patient (proband 2) was found to be homozygous for a G>T transversion in the third base of the ATG translation initiation codon in exon 1, resulting in the conversion of methionine to isoleucine (p.Met1Ile). CONCLUSION: In conclusion, we have identified 2 novel GPIHBP1 missense mutations in 2 unrelated patients as the cause of their severe hypertriglyceridemia.

A 3-day-old neonate with severe hypertriglyceridemia from novel mutations of the GPIHBP1 gene.

BACKGROUND: Familial chylomicronemia is a genetic defect of the intravascular lipolysis of triglyceride (TG)-rich lipoproteins. Intravascular lipolysis involves the TG-hydrolase lipoprotein lipase (LPL) as well as other factors such as apolipoprotein CII and apolipoprotein AV (activators of LPL), GPIHBP1 (the molecular platform required for LPL activity on endothelial surface), and LMF1 (a factor required for intracellular formation of active LPL). METHODS: We sequenced the familial chylomicronemia candidate genes in a neonate with chylomicronemia. RESULTS: A 3-day-old newborn was found to have chylomicronemia (plasma TG 18.8 mmol/L, 1.667 mg/dL). The discontinuation of breastfeeding for 24 hours reduced plasma TG to 2.3 mmol/L (201 mg/dL), whereas its resumption induced a sharp TG increase (7.9 mmol/L, 690 mg/dL). The child was switched to a low-fat diet, which was effective in maintaining TG level below 3.5 mmol/L (294 mg/dL) during the first months of life. The child was found to be a compound heterozygous for 2 novel mutations in GPIHBP1 gene. The first mutation was a 9-bp deletion and 4-bp insertion in exon 2, causing a frameshift that abolished the canonical termination codon TGA. The predicted translation product of the mutant messenger RNA is a peptide that contains 51 amino acids of the N-terminal end of the wild-type protein followed by 252 novel amino acids. The second mutation was a nucleotide change (c.319T>C), causing an amino acid substitution p.(Ser107Pro) predicted in silico to be damaging. CONCLUSIONS: GPIHBP1 mutations should be considered in neonates with chylomicronemia negative for mutations in LPL gene.

Spectrum of mutations of the LPL gene identified in Italy in patients with severe hypertriglyceridemia.

BACKGROUND: Monogenic hypertriglyceridemia (HTG) may result from mutations in some genes which impair the intravascular lipolysis of triglyceride (TG)-rich lipoproteins mediated by the enzyme Lipoprotein lipase (LPL). Mutations in the LPL gene are the most frequent cause of monogenic HTG (familial chylomicronemia) with recessive transmission. METHODS: The LPL gene was resequenced in 149 patients with severe HTG (TG > 10 mmol/L) and 106 patients with moderate HTG (TG > 4.5 and <10 mmol/L) referred to tertiary Lipid Clinics in Italy. RESULTS: In the group of severe HTG, 26 patients (17.4%) were homozygotes, 9 patients (6%) were compound heterozygotes and 15 patients (10%) were simple heterozygotes for rare LPL gene variants. Single or multiple episodes of pancreatitis were recorded in 24 (48%) of these patients. There was no difference in plasma TG concentration between patients with or without a positive history of pancreatitis. Among moderate HTG patients, six patients (5.6%) were heterozygotes for rare LPL variants; two of them had suffered from pancreatitis. Overall 36 rare LPL variants were found, 15 of which not reported previously. Systematic analysis of close relatives of mutation carriers led to the identification of 44 simple heterozygotes (plasma TG 3.2 ± 4.1 mmol/L), none of whom had a positive history of pancreatitis. CONCLUSIONS: The prevalence of rare LPL variants in patients with severe or moderate HTG, referred to tertiary lipid clinics, was 50/149 (33.5%) and 6/106 (5.6%), respectively. Systematic analysis of relatives of mutation carriers is an efficient way to identify heterozygotes who may develop severe HTG.

Severe hypertriglyceridemia in a newborn with monogenic lipoprotein lipase deficiency: an unconventional therapeutic approach with exchange transfusion.

Severe hypertriglyceridemia (sHTG) (plasma triglyceride level > 10 mmol/L) due to lipoprotein lipase (LPL) deficiency is a known risk factor for acute pancreatitis. A 23-day-old male with sHTG was admitted to the Neonatal Intensive Care Unit for plasmapheresis being at high risk for acute pancreatitis. Given the potential hazard of an extracorporeal technique in a very young infant, we decided to perform an exchange transfusion (ET), a procedure widely used by neonatologists and less invasive than plasmapheresis. ET led to a dramatic reduction in plasma triglyceride level, from 93.2 to 3.8 mmol/L at the end of the procedure, without adverse events. The subsequent administration of a special formula low in fat and high in medium-chain triglycerides was effective in keeping fasting plasma triglyceride level below 5.6 mmol/L during the first 5 months of life. The sequence of LPL gene revealed that the patient was apparently homozygous for a novel nucleotide deletion (c.840delG) in exon 6 leading to a premature termination codon (p.N281Mfs*23). However, family studies revealed that while the patient's mother was heterozygous for this mutation, the father was heterozygous for a novel deletion eliminating the whole LPL gene. The patient therefore turned out to be a compound heterozygous for two LPL gene mutations predicted to abolish LPL activity. This is the first case of sHTG treated with ET in a neonate reported in the literature. ET appears to be a safe procedure, alternative to plasmapheresis, to prevent acute pancreatitis in young infants with sHTG due to LPL deficiency.

Can APOE and MTHFR polymorphisms have an influence on the severity of cardiovascular manifestations in Italian Pseudoxanthoma elasticum affected patients?

BACKGROUND: The clinical phenotype of Pseudoxanthoma elasticum (PXE) affected patients, although progressive with age, is very heterogeneous, even in the presence of identical ABCC6 mutations, thus suggesting the occurrence of modifier genes. Beside typical skin manifestations, the cardiovascular (CV) system, and especially the peripheral vasculature, is frequently and prematurely compromised. METHODS AND RESULTS: A cohort of 119 Italian PXE patients has been characterized for apolipoprotein E (APOE) and methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms by PCR. The severity of the clinical phenotype has been quantified according to the Phenodex PXE International score system. Statistical analysis (chi2 test, odd ratio, regression analysis, analysis of variance) were done by GraphPad. Data demonstrate that the frequency of APOE alleles is similar in PXE patients and in healthy subjects and that the allelic variant E2 confers a protection against the age-related increase of CV manifestations. By contrast, PXE patients are characterized by high frequency of the MTHFR-T677T polymorphism. With age, CV manifestations in T677T, but also in C677T, patients are more severe than those associated with the C677C genotype. Interestingly, compound heterozygosity for C677T and A1298C polymorphisms is present in 70% of PXE patients. CONCLUSIONS: PXE patients may be screened for these polymorphisms in order to support clinicians for a better management of disease-associated CV complications.