A novel homozygous AP4B1 mutation in two brothers with AP‐4 deficiency syndrome and ocular anomalies

Adaptor protein complex‐4 (AP‐4) is a heterotetrameric protein complex which plays a key role in vesicle trafficking in neurons. Mutations in genes affecting different subunits of AP‐4, including AP4B1, AP4E1, AP4S1, and AP4M1, have been recently associated with an autosomal recessive phenotype, consisting of spastic tetraplegia, and intellectual disability (ID). The overlapping clinical picture among individuals carrying mutations in any of these genes has prompted the terms “AP‐4 deficiency syndrome” for this clinically recognizable phenotype. Using whole‐exome sequencing, we identified a novel homozygous mutation (c.991C>T, p.Q331*, NM_006594.4) in AP4B1 in two siblings from a consanguineous Pakistani couple, who presented with severe ID, progressive spastic tetraplegia, epilepsy, and microcephaly. Sanger sequencing confirmed the mutation was homozygous in the siblings and heterozygous in the parents. Similar to previously reported individuals with AP4B1 mutations, brain MRI revealed ventriculomegaly and white matter loss. Interestingly, in addition to the typical facial gestalt reported in other AP‐4 deficiency cases, the older brother presented with congenital left Horner syndrome, bilateral optic nerve atrophy and cataract, which have not been previously reported in this condition. In summary, we report a novel AP4B1 homozygous mutation in two siblings and review the phenotype of AP‐4 deficiency, speculating on a possible role of AP‐4 complex in eye development.

     

Putative founder effect of Arg338* AP4M1 (SPG50) variant causing severe intellectual disability,epilepsy and spastic paraplegia: Report of three families

Bi-allelic variants affecting one of the four genes encoding the AP4 subunits are responsible for the “AP4 deficiency syndrome.” Core features include hypotonia that progresses to hypertonia and spastic paraplegia, intellectual disability, postnatal microcephaly, epilepsy, and neuroimaging features. Namely, AP4M1 (SPG50) is involved in autosomal recessive spastic paraplegia 50 (MIM#612936). We report on three patients with core features from three unrelated consanguineous families originating from the Middle East. Exome sequencing identified the same homozygous nonsense variant: NM_004722.4(AP4M1):c.1012C>T p.Arg338* (rs146262009). So far, four patients from three other families carrying this homozygous variant have been reported worldwide. We describe their phenotype and compare it to the phenotype of patients with other variants in AP4M1. We construct a shared single-nucleotide polymorphism (SNP) haplotype around AP4M1 in four families and suggest a probable founder effect of Arg338* AP4M1 variant with a common ancestor most likely of Turkish origin.     

A nonstop variant in REEP1 causes peripheral neuropathy by unmasking a 3′UTR‐encoded,aggregation‐inducing motif

Single‐nucleotide variants that abolish the stop codon (“nonstop” alterations) are a unique type of substitution in genomic DNA. Whether they confer instability of the mutant mRNA or result in expression of a C‐terminally extended protein depends on the absence or presence of a downstream in‐frame stop codon, respectively. Of the predicted protein extensions, only few have been functionally characterized. In a family with autosomal dominant Charcot‐Marie‐Tooth disease type 2, that is, an axonopathy affecting sensory neurons as well as lower motor neurons, we identified a heterozygous nonstop variant in REEP1. Mutations in this gene have classically been associated with the upper motor neuron disorder hereditary spastic paraplegia (HSP). We show that the C‐terminal extension resulting from the nonstop variant triggers self‐aggregation of REEP1 and of several reporters. Our findings support the recently proposed concept of 3′UTR‐encoded “cryptic amyloidogenic elements.” Together with a previous report on an aggregation‐prone REEP1 deletion variant in distal hereditary motor neuropathy, they also suggest that toxic gain of REEP1 function, rather than loss‐of‐function as relevant for HSP, specifically affects lower motor neurons. A search for similar correlations between genotype, phenotype, and effect of mutant protein may help to explain the wide clinical spectra also in other genetically determined disorders.     

Musculocontractural Ehlers‐Danlos Syndrome (former EDS type VIB) and adducted thumb clubfoot syndrome (ATCS) represent a single clinical entity caused by mutations in the dermatan‐4‐sulfotransferase 1 encoding CHST14 gene

We present clinical and molecular findings of three patients with an EDS VIB phenotype from two consanguineous families. The clinical findings of EDS kyphoscoliotic type (EDS type VIA and B) comprise kyphoscoliosis, muscular hypotonia, hyperextensible, thin and bruisable skin, atrophic scarring, joint hypermobility and variable ocular involvement. Distinct craniofacial abnormalities, joint contractures, wrinkled palms, and normal urinary pyridinoline ratios distinguish EDS VIB from EDS VIA. A genome‐wide SNP scan and sequence analyses identified a homozygous frameshift mutation (NM_130468.2:c.145delG, NP_569735.1:p.Val49*) in CHST14, encoding dermatan‐4‐sulfotransferase 1 (D4ST‐1), in two Turkish siblings. Subsequent sequence analysis of CHST14 identified a homozygous 20‐bp duplication (NM_130468.2:c.981_1000dup, NP_569735.1:p.Glu334Glyfs*107) in an Indian patient. Loss‐of‐function mutations in CHST14 were recently reported in adducted thumb–clubfoot syndrome (ATCS). Patients with ATCS present similar craniofacial and musculoskeletal features as the EDS VIB patients reported here, but lack the severe skin manifestations. By identifying an identical mutation in patients with EDS VIB and ATCS, we show that both conditions form a phenotypic continuum. Our findings confirm that the EDS‐variant associated with CHST14 mutations forms a clinical spectrum, which we propose to coin as “musculocontractural EDS” and which results from a defect in dermatan sulfate biosynthesis, perturbing collagen assembly. Hum Mutat 31:1–7, 2010. © 2010 Wiley‐Liss, Inc.     

CD4+CD45RA−FoxP3high activated regulatory T cells are functionally impaired and related to residual insulin‐secreting capacity in patients with type 1 diabetes

Accumulating lines of evidence have suggested that regulatory T cells (T_regs) play a central role in T cell-mediated immune response and the development of type 1A and fulminant type 1 diabetes. CD4⁺forkhead box protein 3 (FoxP3)⁺ T cells are composed of three phenotypically and functionally distinct subpopulations; CD45RA⁺FoxP3^low resting T_regs (r-T_regs), CD45RA⁻FoxP3^high activated T_regs (a-T_regs) and CD45RA⁻FoxP3^low non-suppressive T cells (non-T_regs). We aimed to clarify the frequency of these three subpopulations in CD4⁺FoxP3⁺ T cells and the function of a-T_regs with reference to subtypes of type 1 diabetes. We examined 20 patients with type 1A diabetes, 15 patients with fulminant type 1 diabetes, 20 patients with type 2 diabetes and 30 healthy control subjects. A flow cytometric analysis in the peripheral blood was performed for the frequency analysis. The suppressive function of a-T_regs was assessed by their ability to suppress the proliferation of responder cells in a 1/2:1 co-culture. A flow cytometric analysis in the peripheral blood demonstrated that the frequency of a-T_regs was significantly higher in type 1A diabetes, but not in fulminant type 1 diabetes, than the controls. Further, the proportion of a-T_regs among CD4⁺FoxP3⁺ T cells was significantly higher in patients with type 1A diabetes with detectable C-peptide but not in patients with type 1A diabetes without it and with fulminant type 1 diabetes. A proliferation suppression assay showed that a-T_regs were functionally impaired both in fulminant type 1 diabetes and in type 1A diabetes. In conclusion, a-T_regs were functionally impaired, related to residual insulin-secreting capacity and may be associated with the development of type 1 diabetes.     

Identification and functional analysis of an ADAMTSL1 variant associated with a complex phenotype including congenital glaucoma,craniofacial, and other systemic features in a three‐generation human pedigree

Developmental glaucoma can occur as an isolated or syndromic condition and is genetically heterogeneous. We describe a three‐generation family affected with developmental glaucoma, myopia, and/or retinal defects associated with variable craniofacial/dental, auditory, brain, renal, and limb anomalies. Whole‐exome sequencing identified a heterozygous c.124T> C, p.(Trp42Arg) allele in ADAMTSL1; cosegregation analysis confirmed the presence of this allele in four affected family members. The mutation affects a highly conserved residue and is strongly predicted to have a deleterious effect on protein function. Trp42 is normally modified by protein C‐mannosylation, an unusual post‐translational modification. Comparison of ADAMTSL1‐WT (also known as punctin‐1) and ADAMTSL1‐p.Trp42Arg in vitro demonstrated that the latter was not secreted from transfected cells but retained intracellularly. Moreover, ADAMTSL1‐p.Trp42Arg reduced secretion of cotransfected wild‐type ADAMTSL1, suggesting a dominant negative effect for this mutation. These data imply a multisystem role for ADAMTSL1 and present the first disease‐associated variant affecting a C‐mannosylation motif.     

The PTPN22 1858T allele but not variants in the proximal promoter region of IL‐21 gene is associated with the susceptibility to type 1 diabetes and the presence of autoantibodies in a Brazilian cohort

Interleukin (IL)-21 and protein tyrosine phosphatase non-receptor 22 (PTPN22) regulate lymphocyte function and have been implicated in the pathogenesis of autoimmune diabetes. We sequenced the proximal promoter of the IL-21 gene for the first time and analysed the PTPN22 1858T polymorphism in type 1A diabetes (T1AD) patients and healthy controls (HC). We correlated the frequencies of islet and extra-pancreatic autoantibodies with genotypes from both loci. The case series comprised 612 T1AD patients and 792 HC. Genotyping of PTPN22 C1858T was performed on 434 T1AD patients and 689 HC. The −448 to +83 base pairs (bp) region of the IL-21 gene was sequenced in 309 Brazilian T1AD and 189 HC subjects. We also evaluated human leucocyte antigen (HLA) DR3/DR4 alleles. The frequencies of glutamic acid decarboxylase (GAD65), tyrosine phosphatase-like protein (IA)-2, anti-nuclear antibody (ANA), thyroid peroxidase (TPO), thyroglobulin (TG), thyrotrophin receptor autoantibody (TRAb), anti-smooth muscle (ASM) and 21-hydroxylase (21-OH) autoantibodies were higher in T1AD patients than in HC. The PTPN22 1858T allele was associated with an increased risk for developing T1AD [odds ratio (OR) = 1·94; P < 0·001], particularly in patients of European ancestry, and with a higher frequency of GAD65 and TG autoantibodies. HLA-DR3/DR4 alleles predominated in T1AD patients. A heterozygous allelic IL-21 gene variant (g.-241 T > A) was found in only one patient. In conclusion, only PTPN22 C1858T polymorphism and HLA-DR3 and/or DR4 alleles, but not allelic variants in the 5′-proximal region of the IL-21 gene were associated with T1AD risk. Patients with T1AD had increased frequencies of anti-islet-cell, anti-thyroid, anti-nuclear, anti-smooth muscle and anti-21-OH autoantibodies. The C1858T PTPN22 polymorphism was also associated with a higher frequency of GAD65 and TG autoantibodies.