Rapid genetic diversification within dog breeds as evidenced by a case study on Schnauzers.

As a result of strong artificial selection, the domesticated dog has arguably become one of the most morphologically diverse vertebrate species, which is mirrored in the classification of around 400 different breeds. To test the influence of breeding history on the genetic structure and variability of today's dog breeds, we investigated 12 dog breeds using a set of 19 microsatellite markers from a total of 597 individuals with about 50 individuals analysed per breed. High genetic diversity was noted over all breeds, with the ancient Asian breeds (Akita, Chow Chow, Shar Pei) exhibiting the highest variability, as was indicated chiefly by an extraordinarily high number of rare and private alleles. Using a Bayesian clustering method, we detected significant genetic stratification within the closely related Schnauzer breeds. The individuals of these three recently differentiated breeds (Miniature, Standard and Giant Schnauzer) could not be assigned to a single cluster each. This hidden genetic structure was probably caused by assortative mating owing to breeders' preferences regarding coat colour types and the underlying practice of breeding in separate lineages. Such processes of strong artificial disruptive selection for different morphological traits in isolated and relatively small lineages can result in the rapid creation of new dog types and potentially new breeds and represent a unique opportunity to study the evolution of genetic and morphological differences in recently diverged populations.

[Inclusion body myositis, Paget's disease of the bone and frontotemporal dementia: early involvement of the heart and respiratory muscles]. / Inclusion Body Myositis mit Morbus Paget und frontotemporaler Demenz: frühe Beteiligung des Herzens und der Atemmuskulatur.

Since valosin-containing protein mutations were reported as a cause of hereditary inclusion body myositis associated with Paget's disease of the bone and frontotemporal dementia, many new mutations have been described in the last decade. We report on a 46-year-old German male with a progressive tetraparesis and autosomal dominant inheritance pattern. Echocardiography revealed a beginning dilated cardiomyopathy and laboratory analyses showed increased alkaline phosphatase. Decreased verbal memory and an impairment of concept building were observed on neuropsychological examination. Muscle biopsy demonstrated a myopathic pattern, rimmed vacuoles, CD8+ T-cell infiltrates and positive MHC1-muscle fibres. We found a heterozygote mutation in exon 5 of the valosin-containing protein gene (c.464G > T p.Arg155Leu), which until now has been described only in an Australian family. We describe here the first German case with the above-mentioned mutation causing inclusion-body myositis associated with Paget's disease of the bone and fronto-temporal dementia. Here, we recommend regular controls of cardiac and respiratory functions.

Tracing the origin of 'blue Weimaraner' dogs by molecular genetics.

Weimaraner dogs are defined by light brown coat colour termed grey including several shadings ranging from silver and deer to mouse grey. In contrast, the so-called blue Weimaraners (BW) with lightened black-pigmented coat have been proposed to represent spontaneous revertants in the Weimaraner breed. In order to investigate the genetic determinants of the characteristic grey coat colour versus those of BW, known variation in coat colour genes including TYRP1 and MLPH were analysed in a number of grey and blue dogs. Variations at the B locus cause grey coat colour in Weimaraners via two non-functional TYRP1 copies (bb) including the b(s), b(d) and b(c) alleles. In all BW, at least one functional TYRP1 allele (Bb or BB genotype) was identified. Defined microsatellite alleles in TYRP1 intron 4 are linked to this functional B allele in BW. These alleles were also detected in various other dog breeds, but not in grey Weimaraners. The combination of a dominant trait for blue versus grey together with a specific TYRP1 haplotype in BW suggests that blue coat colour is not the result of spontaneous (back-) mutation in grey Weimaraners. This inference is even emphasized by the presence of a unique Y-chomosomal haplotype in a male offspring of the supposed ancestor of the BW population which - according to pedigree information - carries a copy of the original Y chromosome. Thus, molecular genetic analyses of coat colours combined with Y-chromosomal haplotypes allow tracing the origin of atypical dogs in respective canine populations.

On ancestors of dog breeds with focus on Weimaraner hunting dogs.

Paternally inherited Y chromosomal markers and maternally inherited mitochondrial (mt) DNA sequences were investigated in 27 dog breeds (Canis familiaris), of which the Weimaraner hunting dog was studied in greater detail. Altogether, nine potentially polymorphic markers of the Y chromosome were examined as well as parts of the canine mt genome (1947 base pairs) in 111 male dogs and four wolves for comparison. Twenty Y chromosomal and fifty-nine mitochondrial DNA (mtDNA) haplotypes were identified in the canine breeds and wolves. In 34 Weimaraners, four distinct Y chromosomal haplotypes were observed as well as three mtDNA types thus reflecting at least four male and three female ancestors for the current population in Germany. Tracing patri- and matrilineages, several entries in the Weimaraner stud book cannot be reconciled with the male-only, Y chromosomal neither the female-only, mt inheritance patterns, respectively. The investigated breeds represent 9 of 10 groups defined by the Fédération Cynologique Internationale (FCI). The level of Y chromosomal and especially mtDNA diversity was immense considering the relatively small number of individuals investigated per breed. Unique haplotypes were found only in a few breeds and the wolf. Other haplotypes were shared among several breeds, also across different FCI groups, suggesting that these canine breeds had common male and female ancestors.

New cases of adult-onset Sandhoff disease with a cerebellar or lower motor neuron phenotype.

Sandhoff disease is a lipid-storage disorder caused by a defect in ganglioside metabolism. It is caused by a lack of functional N-acetyl-beta-d-glucosaminidase A and B due to mutations in the HEXB gene. Typical, early-onset Sandhoff disease presents before 9 months of age with progressive psychomotor retardation and early death. A late-onset form of Sandhoff disease is rare, and its symptoms are heterogeneous. As drug trials that aim to intervene in the disease mechanism are emerging, the recognition and identification of Sandhoff disease patients-particularly those with atypical phenotypes-are becoming more important. The authors describe six new late-onset Sandhoff cases demonstrating cerebellar ataxia or lower motor neuron (LMN) involvement combined with, mostly subclinical, neuropathy. Two different mutations were found: IVS 12-26 G/A and c.1514G-->A. In patients with either progressive cerebellar ataxia or LMN disease in the setting of a possibly recessive disorder, Sandhoff disease should be suspected, even when the onset age is over 45 years.

Substrate deprivation therapy in juvenile Sandhoff disease.

Substrate deprivation therapy has been successfully applied in a number of lysosomal storage diseases, such as Gaucher disease. So far only limited experience is available in Sandhoff disease. We initiated substrate deprivation therapy in one male patient, who initially presented at the age of 3.5 years with epilepsy and regression in motor skills and speech development. Juvenile Sandhoff disease was diagnosed on the basis of a decreased hexosaminidase activity in leukocytes and a homozygous HEXB gene mutation. After the epilepsy was controlled, the clinical course remained stable for years, defined by a mild proximal myopathy and stable mental retardation. At 14 years of age the patient experienced a second episode with progressively worsening general condition with diminishing muscle power and progressive ataxia. Treatment was started with the N-alkylated imino sugar miglustat, inhibiting the glucosylceramide synthase, an essential enzyme for the synthesis of glycosphingolipids. Diarrhoea was treated with lactose restriction. We performed detailed biochemical investigations, motor and mental development analysis, brain imaging, organ function studies and quality of life score prior to and at different time points after start of the treatment. Two years after the initiation of therapy the patient has a stable neurological picture without further regression in his motor development, ataxia or intelligence. There is a subjective improvement in the fine motor skills and walking up the stairs but no change in the quality of life score. Under treatment with miglustat the clinical course in our patient with Sandhoff disease did not further deteriorate.

Pancreatitis in primary hyperparathyroidism-related hypercalcaemia is not associated with mutations in the CASR gene.

BACKGROUND: Primary hyperparathyroidism (pHPT) related hypercalcaemia is considered to represent a risk factor for the development of pancreatitis. We therefore explored whether mutations in the calcium-sensing receptor gene ( CASR) coding for the calcium-sensing receptor (CaR), an essential regulator of the calcium homeostasis in parathyroid chief cells, exist in a cohort of patients with pHPT and pancreatitis. METHODS: Among 826 patients prospectively studied between 1987 and 2002 with pHPT, 38 patients were identified with pancreatitis (4.6%). DNA was available of 25 patients (13 females and 12 males). These individuals were analysed for mutations in the CASR by single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. RESULTS: None of the 25 patients with pHPT and pancreatitis carried a CASR mutation and only one had a known heterozygous polymorphism R990G. CONCLUSIONS: Pancreatitis in primary hyperparathyroidism is not associated with mutations in the CASR gene, while it remains to be determined why the polymorphisms A986S, R990G and Q1011E were less often present in that subgroup than in the normal population.

Alpha vs. gamma sarcoglycanopathy: DNA tests solve a case from Argentina.

Immunohistochemical and DNA results are described in a patient with sarcoglycanopathy. Immunostaining was comparatively normal for alpha-, attenuated for beta- and delta-, and markedly attenuated for gamma-sarcoglycan, thus sarcoglycanopathy was diagnosed, presumably a gamma-sarcoglycanopathy. Unexpectedly, two alpha-SGP-related pathogenic mutations were identified in compound heterozygosity in the SGCA gene: c.229C > T (p.Arg77Cys) in exon 3 and c.850C > T (p.Arg284Cys) in exon 7. These are discussed together with six additional changes detected in SGCB, SGCG and SGCD.

Respiratory insufficiency as a presenting symptom of LGMD2D in adulthood.

Several forms of recessive limb girdle muscular dystrophy (LGMD2C-F) are due to mutations in genes coding for sarcoglycans. Clinically, most sarcoglycanopathies present in childhood with skeletal muscle wasting and early loss of ambulation; respiratory insufficiency is rare. However, some cases of LGMD2D with a late onset and a milder course have been reported. In this study, two adult brothers, compound heterozygous for two missense mutations of the SGCA gene (Arg77Cys, Val247Met), presented with respiratory insufficiency while they were still ambulatory.

Autosomal dominant nemaline myopathy caused by a novel alpha-tropomyosin 3 mutation.

Nemaline myopathy (NM) is a genetically and clinically heterogenous muscle disorder, which is myopathologically characterized by nemaline bodies. Mutations in six genes have been reported to cause NM: Nebulin (NEB Pelin 1999), alpha-skeletal muscle actin (ACTA1 Nowak 1999), alpha-slow tropomyosin (TPM3 Laing 1995), beta-tropomyosin (TPM2 Donner 2002), slow troponin T (TNNT1 Johnston 2000) and cofilin 2 (CFL2 Agrawal 2007). The majority of cases are due to mutation in NEB and ACTA1. We report on the clinical, myopathological and muscle MRI findings in a German family with autosomal dominant NM due to a novel pathogenic TPM3 mutation (p.Ala156Thr).

Identification of a tyrosinase (TYR) exon 4 deletion in albino ferrets (Mustela putorius furo).

Albinism is due to a lack of pigmentation in hair, skin and eye, and has been shown to occur in several animal species. Mutations of the tyrosinase (TYR) gene account for albinism in domestic cats, rabbits, cattle, mice and rats. In this study, we demonstrate that a TYR mutation accounts for albinism in the ferret (Mustela putorius furo). The coding sequence of the five exons of TYR was determined in genomic DNA from wild-type pigmented 'sable' coloured and albino ferrets. It was not possible to amplify TYR exon 4 in albino ferrets originating from different breeds. The deletion of exon 4 in albino ferrets was confirmed by Southern blot hybridization of genomic DNA from albino and pigmented ferrets. This is the first report of a deletion of a TYR exon in a non-human mammal.

Mutations in the calcium-sensing receptor: a new genetic risk factor for chronic pancreatitis?

OBJECTIVE: In 2003 we identified a family with familial hypocalciuric hypercalcemia (FHH) (heterozygous CASR gene mutation L173P) and a mutation in the pancreatic secretory trypsin inhibitor gene (SPINK1) (N34S). While family members with an isolated calcium-sensing receptor gene (CASR) mutation remained healthy, a combination of the CASR and SPINK1 gene mutation caused chronic pancreatitis (CP). We thus speculate that the combination of two genetic defects affecting calcium homeostasis and pancreatic enzyme activation might represent a novel approach in chronic inherited pancreatic disease. We therefore sought to explore whether CASR gene mutations were prevalent in a cohort of patients with CP and confirmed SPINK1 mutations. MATERIAL AND METHODS: A cohort of 19 families (n=170) with a history of idiopathic CP (ICP) was screened for mutations within the CASR gene; 104 members of that cohort had a mutation (N34S) within the SPINK1 gene and 66 of those were suffering from CP. The entire CASR gene was screened for single strand conformation polymorphism under varying polyacrylamide gel conditions and subjected to direct dideoxy nucleotide sequencing of amplified cDNA. RESULTS: Single-strand conformation polymorphisms were observed in 59 samples, clustering of exons 3, 4 and 7. DNA sequence analysis revealed a yet unreported missense mutation in exon 7 (R896H) and two conservative mutations in exon 4 (F391F) and exon 7 (E790E). Furthermore, an intronic polymorphism in nucleotide position 493-19 G>A was detected in 19 out of 170 members of that cohort. CONCLUSIONS: We identified three novel calcium-sensing receptor gene mutations (1 missense mutation, 2 silent mutations and 1 intronic polymorphism) in a cohort of 19 families with ICP. In particular, the kindred with the R896H mutation presenting with a similar pedigree to the family described above may indicate a role for CASR gene mutations in SPINK1-related CP. Again, only the patient with the combination of both CASR and N34S SPINK1 gene mutation developed pancreatitis, whereas in the healthy parents and children only an isolated CASR or N34S SPINK1 gene mutation could be detected. We suggest that the CASR gene is a novel yet undetected co-factor in a multifactorial genetic setting of SPINK1-related pancreatitis that alters the susceptibility for pancreatitis in these patients.

Parkin gene variations in late-onset Parkinson's disease: comparison between Norwegian and German cohorts.

OBJECTIVES: Mutations in the Parkin gene can cause autosomal recessive early-onset Parkinson's disease (PD). Recently, Parkin mutations were also suggested to play a role in the commoner late-onset forms of PD. METHODS: We compared a German cohort of PD patients (95) with a Norwegian cohort of PD patients (96). Both cohorts have predominant late-onset form of PD. Mutation and polymorphism frequencies were compared via single-strand conformation polymorphism and sequence analyses. RESULTS: Three heterozygous missense mutations (Arg256Cys, Arg402Cys and Thr240Met) were found in late-onset PD patients in the German patient cohort (1.6%). A missense mutation (Arg402Cys) was also found in one of 149 healthy control subjects (0.3%). Only one heterozygous missense mutation (Arg256Cys) was identified in a Norwegian patient suffering from late-onset PD (0.5%). The frequencies of four known single nucleotide polymorphisms significantly differ between the two distant European populations. CONCLUSION: The results support the hypothesis that heterozygous mutations in the Parkin gene may act as susceptibility alleles for late-onset forms of PD in rare cases.

Exclusion of the PDE6A gene for generalised progressive retinal atrophy in 11 breeds of dog.

The cyclic guanosine monophosphate specific phosphodiesterase (cGMP-specific PDE) is a key enzyme in the phototransduction cascade of the vertebrate retina. This enzyme consists of two catalytic alpha and beta subunits, two identical inhibitory gamma subunits as well as a delta subunit. Mutations in PDE6A and the PDE6B genes lead to autosomal recessive (ar) forms of retinitis pigmentosa (RP) in human and to the homologous disease in dogs, designated generalised progressive retinal atrophy (gPRA). We investigated the PDE6A gene in 13 gPRA-affected dog breeds including healthy animals, obligate gPRA carriers and gPRA-affected dogs. In the coding region of PDE6A only a rare sequence variation (G103A; Asp35Asn) was found in exon 1 of two healthy Tibet Terriers and one affected Cocker Spaniel. Using single-stranded conformation polymorphism (SSCP) analyses we detected several sequence variations in eight of the PDE6A introns in different investigated breeds. Most informative for excluding the PDE6A gene as a cause for gPRA was a polymorphic microsatellite ((GT)10CG(GT)2CG(GT)12) in intron 14 and four sequence variations in intron 18 for almost all breeds investigated. The sequence variations of PDE6A did not segregate together with gPRA in 11 breeds. Since diseased animals were heterozygous for the polymorphisms, the PDE6A gene is unlikely to harbour the critical mutation causing gPRA in the following breeds: Chesapeake Bay Retriever. Entlebucher Sennenhund, Labrador Retriever. Tibet Mastiff, Dachshund (long- and wire-haired), Tibetan Terrier, Miniature Poodle. Australian Cattle Dog, Cocker Spaniel, Saarloos/Wolfshound, Sloughi.

Evaluation of RDS/Peripherin and ROM1 as candidate genes in generalised progressive retinal atrophy and exclusion of digenic inheritance.

Generalised progressive retinal atrophy (gPRA) is a heterogeneous group of hereditary diseases causing degeneration of the retina in dogs and cats. As a combination of mutations in the RDS/Peripherin and the ROM1 genes leads to the phenotype of retinitis pigmentosa in man we first performed mutation analysis to screen these genes for disease causing mutations followed by the investigation of a digenic inheritance in dogs. We cloned the RDS/Peripherin gene and investigated the RDS/Peripherin and ROM1 genes for disease causing mutations in 13 gPRA-affected dog breeds including healthy animals, obligate gPRA carriers and gPRA-affected dogs. We screened for mutations using single strand conformation polymorphism (SSCP) analysis. Sequence analysis revealed several sequence variations. In the coding region of the RDS/Peripherin gene three nucleotide exchanges were identified (A277C; C316T; G1255A), one of which leads to an amino acid substitution (Ala339Thr). Various silent sequence variations were found in the coding region of the ROM1 gene (A536G, G1006A, T1018C, T1111C, C1150T, C1195T), as well as an amino acid substitution (G252T; Ala54Ser). By excluding the respective gene as a cause for gPRA several sequence variations in the intronic regions were investigated. None of these sequence variations cosegregated with autosomal recessively (ar) transmitted gPRA in 11 breeds. The candidate gene RDS/Peripherin obviously does not harbour the critical mutation causing the autosomal recessive form of gPRA because diseased individuals show heterozygous genotypes for sequence variations in the Miniature Poodle, Dachshund, Australian Cattle Dog, Cocker Spaniel, Chesapeake Bay Retriever, Entlebucher Sennenhund, Sloughi, Yorkshire Terrier, Tibet Mastiff, Tibet Terrier and Labrador Retriever breeds. In the following breeds the ROM1 gene was also excluded indirectly for gPRA: Miniature Poodle, Dachshund, Australian Cattle Dog, Sloughi, Collie, Tibet Terrier, Labrador Retriever and Saarloos/Wolfhound. Digenic inheritance for gPRA is practically excluded for both these genes in four breeds: Miniature Poodle, Dachshund, Labrador Retriever and Saarloos/Wolfhound.

Generalized progressive retinal atrophy of Sloughi dogs is due to an 8-bp insertion in exon 21 of the PDE6B gene.

We investigated the gene encoding the beta subunit of cGMP phosphodiesterase (PDE6B) as a candidate for generalized progressive retinal atrophy (gPRA), an autosomal recessively transmitted eye disease in dogs. The PDE6B gene was isolated from a genomic library. Single-strand conformation polymorphism analysis revealed eight intronic variations in different subsets of the 14 dog breeds investigated. In addition, we identified an 8-bp insertion after codon 816 in certain Sloughi dogs. Analysis of PRA-affected and obligatory carrier Sloughis showed that this mutation cosegregates with disease status in a large pedigree. All other exchanges identified were not located in functionally relevant parts of the gene (e.g., in the splice signal consensus sites). In most dog breeds (Labrador retriever, Tibetan mastiff, dachshund, Tibetan terrier, miniature poodle, Australian cattle dog, cocker spaniel, collie, Saarloos wolfhound, Chesapeake Bay retriever, and Yorkshire terrier), PDE6B was excluded as a candidate gene for gPRA because heterozygous allele constellations were detected in diseased animals. Therefore, the PDE6B sequence variations did not segregate together with the mutation(s) causing gPRA. Direct and indirect DNA tests concerning gPRA can be offered now for a variety of different dog breeds.

Evaluation of ROM1 as a candidate gene in generalised progressive retinal atrophy in dogs.

Generalised progressive retinal atrophy (gPRA) is a heterogeneous group of hereditary diseases causing degeneration of the retina in dogs and other animals. The genetic origin is unknown in most cases. We have screened the coding sequence of the ROM1 gene for disease causing mutations in Tibetan Terriers, Miniature Poodles, Dachshunds and Chesapeake Bay Retrievers by single strand conformation polymorphism analysis (SSCP). Two polymorphisms have been identified by sequencing, one in exon 1 in all examined breeds (position 210: G-->A; Gly40Arg and position 252: G-->T; Ala53-Ser). Another polymorphism was present in exon 2 (position 1150: C-->T and position 1195: C-->T) segregating in Miniature Poodles. None of these polymorphisms were cosegregating with gPRA rendering a disease causing mutation in the ROM1 gene unlikely.