AP1S1 missense mutations cause a congenital enteropathy via an epithelial barrier defect.

Congenital diarrheal disorders (CDD) comprise > 50 monogenic entities featuring chronic diarrhea of early-onset, including defects in nutrient and electrolyte absorption, enterocyte polarization, enteroendocrine cell differentiation, and epithelial integrity. Diarrhea is also a predominant symptom in many immunodeficiencies, congenital disorders of glycosylation, and in some defects of the vesicular sorting and transporting machinery. We set out to identify the etiology of an intractable diarrhea in 2 consanguineous families by whole-exome sequencing, and identified two novel AP1S1 mutations, c.269T>C (p.Leu90Pro) and c.346G>A (p.Glu116Lys). AP1S1 encodes the small subunit of the adaptor protein 1 complex (AP-1), which plays roles in clathrin coat-assembly and trafficking between trans-Golgi network, endosomes and the plasma membrane. An AP1S1 knock-out (KO) of a CaCo2 intestinal cell line was generated to characterize intestinal AP1S1 deficiency as well as identified mutations by stable expression in KO background. Morphology and prototype transporter protein distribution were comparable between parental and KO cells. We observed altered localization of tight-junction proteins ZO-1 and claudin 3, decreased transepithelial electrical resistance and an increased dextran permeability of the CaCo2-AP1S1-KO monolayer. In addition, lumen formation in 3D cultures of these cells was abnormal. Re-expression of wild-type AP1S1 in CaCo2-AP1S1-KO cells reverted these abnormalities, while expression of AP1S1 containing either missense mutation did not. Our data indicate that loss of AP1S1 function causes an intestinal epithelial barrier defect, and that AP1S1 mutations can cause a non-syndromic form of congenital diarrhea, whereas 2 reported truncating AP1S1 mutations caused MEDNIK syndrome, characterized by mental retardation, enteropathy, deafness, neuropathy, ichthyosis, and keratodermia.

Congenital secretory diarrhoea caused by activating germline mutations in GUCY2C.

OBJECTIVE: Congenital sodium diarrhoea (CSD) refers to a form of secretory diarrhoea with intrauterine onset and high faecal losses of sodium without congenital malformations. The molecular basis for CSD remains unknown. We clinically characterised a cohort of infants with CSD and set out to identify disease-causing mutations by genome-wide genetic testing. DESIGN: We performed whole-exome sequencing and chromosomal microarray analyses in 4 unrelated patients, followed by confirmatory Sanger sequencing of the likely disease-causing mutations in patients and in their family members, followed by functional studies. RESULTS: We identified novel de novo missense mutations in GUCY2C, the gene encoding receptor guanylate cyclase C (GC-C) in 4 patients with CSD. One patient developed severe, early-onset IBD and chronic arthritis at 4 years of age. GC-C is an intestinal brush border membrane-bound guanylate cyclase, which functions as receptor for guanylin, uroguanylin and Escherichia coli heat-stable enterotoxin. Mutations in GUCY2C were present in different intracellular domains of GC-C, and were activating mutations that enhanced intracellular cyclic guanosine monophosphate accumulation in a ligand-independent and ligand-stimulated manner, following heterologous expression in HEK293T cells. CONCLUSIONS: Dominant gain-of-function GUCY2C mutations lead to elevated intracellular cyclic guanosine monophosphate levels and could explain the chronic diarrhoea as a result of decreased intestinal sodium and water absorption and increased chloride secretion. Thus, mutations in GUCY2C indicate a role for this receptor in the pathogenesis of sporadic CSD.

Mutations in SPINT2 cause a syndromic form of congenital sodium diarrhea.

Autosomal-recessive congenital sodium diarrhea (CSD) is characterized by perinatal onset of a persistent watery diarrhea with nonproportionally high fecal sodium excretion. Defective jejunal brush-border Na(+)/H(+) exchange has been reported in three sporadic patients, but the molecular basis of the disease has not been elucidated. We reviewed data from a large cohort of CSD patients (n = 24) and distinguished CSD associated with choanal or anal atresia, hypertelorism, and corneal erosions--i.e., a syndromic form of CSD--occurring in ten families from an isolated form--i.e., classic CSD--presenting in seven families. Patients from both groups have a high risk of mortality due to immediate electrolyte imbalances and complications from long-term parenteral nutrition in the first years of life, but survivors can eventually adapt to partial or complete enteral nutrition. A genome-wide SNP scan was applied and identified a homozygous c.593-1G-->A splicing mutation in SPINT2, encoding a Kunitz-type serine-protease inhibitor, in one extended kindred with syndromic CSD. The same mutation and four distinct, homozygous or compound heterozygous mutations (p.Y163C, c.1A-->T, c.337+2T-->C, c.553+2T-->A) were identified in all syndromic patients. No SPINT2 mutations were found in classic-CSD patients. SPINT2 mutations were associated with loss of protein synthesis or failure to inhibit the serine protease trypsin in vitro. We delineate syndromic CSD as a distinct disease entity caused by SPINT2 loss-of-function mutations. SPINT2 mutations might lead to an excess of yet unknown serine protease activity in affected tissues.

On transient breastfeeding-related chylomicronaemia syndrome.

UNLABELLED: This paper reports on two male infants with abnormally high levels of serum triacylglycerols (>15.00 mmol/l) and massive accumulation of chylomicrons. Pathological lipidograms were observed during breastfeeding only and were typical of a rare chylomicronaemia syndrome. Laboratory abnormalities were detected accidentally in the otherwise healthy infants. An unrecognized modulating factor in fresh mother's milk caused transitory decreased activity of hydrolytic complex for chylomicrons and very-low-density lipoproteins, probably due to a dysfunction of lipoprotein lipase. The normalization of lipidograms suggested that the catalytic activity of lipoprotein lipase rapidly recovered after weaning. Pathogenesis moved more towards an immune disorder, to the production of (auto)antibodies against a component of the lipolytic system. Mother's milk should be substituted with banked donor human milk, but this is not an unambiguous demand. CONCLUSION: Fresh mother's milk caused a decrease in the activity of lipolytic enzyme (lipoprotein lipase). The massive hypertriacylglycerolaemia quickly disappeared after weaning. Two infants with this transient chylomicronaemia syndrome were asymptomatic.

Analbuminemia in a Slovak Romany (gypsy) family: case report and mutational analysis.

BACKGROUND: Analbuminemia is a rare autosomal recessive disorder manifested by the absence, or severe reduction, of circulating serum albumin. Here we report three new cases of hereditary analbuminemia, fortuitously detected in three Slovak Romany children, members of the same family, and define the molecular defect that causes the analbuminemic trait. METHODS: Total DNA, extracted from peripheral blood samples from six members of the family, was PCR-amplified using oligonucleotide primers designed to amplify the 14 exons of the human albumin gene and the flanking intron regions. The products were screened for mutations by single-strand conformation polymorphism (SSCP) and heteroduplex analyses (HA). HA allowed the identification of the abnormal fragment, which was then sequenced. RESULTS: In the 3 patients the analbuminemic trait was caused by the same mutation, an AT deletion at nucleotides 2430-31, the 91 th and 92 th bases of exon 3. This defect, previously identified as Kayseri mutation, produces a frameshift leading to a premature stop, two codons downstream. The predicted translation product would consist of 54 amino acid residues. The parents were found to be heterozygous for the mutation. CONCLUSIONS: Our results confirm that the combination of SSCP and HA represents a powerful tool to study the molecular defects causing analbuminemia in humans.

Genetic variants of ApoE account for variability of plasma low-density lipoprotein and apolipoprotein B levels in FHBL.

We report two novel APOB mutations causing short apolipoprotein B (apoB) truncations undetectable in plasma and familial hypobetalipoproteinemia (FHBL). In Family 56, a 5 bp deletion in APOB exon 7 (870_874del5) causes a frame shift, converting tyrosine to a stop codon (Y220X) and producing an apoB-5 truncation. In Family 59, a point mutation (1941G>T) in APOB exon 13 converts glutamic acid to stop codon (E578X), specifying apoB-13. A recurrent mutation in exon 26 (4432delT) produces apoB-30.9 in Family 58. In some members of these families, we observed that plasma low-density lipoprotein (LDL) cholesterol and apoB levels were unusually low even for subjects heterozygous for FHBL. To ascertain whether genetic variations in apolipoprotein E (apoE) would explain some of the variations of apoB and LDL cholesterol levels, apoE genotypes were assessed in affected subjects from a total of eight FHBL families with short apoB truncations. Heterozygous FHBL with the epsilon3/epsilon4 genotype had 10-1 5mg/dL higher plasma LDL cholesterol and apoB levels compared to subjects with the epsilon2/epsilon3 and epsilon3/epsilon3 genotypes. The apoE genotype has been reported to account for approximately 10% of the variation of LDL cholesterol in the general population. It accounted for 15-60% of the variability of plasma LDL cholesterol or apoB levels in our FHBL subjects. The physiologic bases for the greater effects of apoE in FHBL remain to be determined.