WO2013037371A1

WO2013037371A1 - Genetic variants associated with infantile hypertrophic pyloric stenosis

Info

Publication number: WO2013037371A1
Application number: PCT/DK2012/000100
Authority: WO
Inventors: Bjarke FEENSTRA; Frank Geller; Mads Melbye
Original assignee: Statens Serum Institut
Priority date: 2011-09-13
Filing date: 2012-09-12
Publication date: 2013-03-21

Abstract

The present invention relates to an in vitro method for diagnosing a genetic predisposition or susceptibility for Infantile Hypertrophic Pyloric Stenosis (IHPS), a severe condition characterized by hypertrophy of the pyloric sphincter muscle. The present invention also relates to diagnostic and research kits for use in diagnosing IHPS and to the use of cholesterol in the treatment of IHPS.

Description

Genetic variants associated with Infantile Hypertrophic Pyloric Stenosis

Field of the invention

The present invention relates to an in vitro method for diagnosing a genetic predisposition or susceptibility for Infantile Hypertrophic Pyloric Stenosis (IHPS), a 5 severe condition characterized by hypertrophy of the pyloric sphincter muscle. The present invention also relates to diagnostic and research kits for use in diagnosing IHPS and to the use of cholesterol in the treatment of IHPS.

Background

10 Infantile Hypertrophic Pyloric Stenosis (IHPS) is a severe condition of infancy in which hypertrophy of the pyloric sphincter smooth muscle leads to obstruction of the gastric-outlet. Symptoms typically appear between 2 and 8 weeks after birth ¹ and include non-bilious projectile vomiting that progress to dehydration, weight loss and hypochloremic, hypokalemic metabolic alkalosis.

15

Treatment may require correction of electrolyte disturbances and curative surgical incision of the pyloric sphincter muscle, a safe and effective procedure introduced by Ramstedt ² a century ago. Because vomiting is common in young infants the diagnosis may be delayed and can result in significant morbidity and mortality 20 when access to appropriate care is limited.

The incidence of IHPS among whites is 1.5 to 3 per 1000 live births ^3, and it is the most common condition requiring surgery in the first months of life. There is a pronounced male excess in the incidence of IHPS, with affected boys

25 outnumbering girls in a 4 to 1 ratio^{5, 6}.

While environmental risk factors such as exposure to erythromycin and bottle feeding have been reported, IHPS also aggregates strongly in families ⁶. In a population-based cohort study of almost 2 million children, there was a nearly 30 200-fold increased risk in monozygotic twins, a 20-fold increased risk among

siblings and with heritability estimated to be 87%⁴. IHPS is generally regarded as a complex disease with multiple genetic and environmental factors contributing to disease pathogenesis^{3, 7}.

Concerning the pathophysiology of IHPS, it has been hypothesized that strong, prolonged contractions of the pyloric sphincter muscle induce hypertrophy of the muscle layer eventually resulting in gastric outlet obstruction. Factors affecting gastric contractility are therefore believed to be central to the development of the disease. Several studies suggest that early exposure to erythromycin in high doses causes IHPS through such a mechanism^{8, 9}. Being a motilin agonist, erythromycin specifically induces excessive pyloric/gastric motility. Another line of studies have investigated the role of neuronal nitric oxide synthase (nNOS), an enzyme that catalyzes the formation of nitric oxide in neurons of the enteric , nervous system. Nitric oxide mediates relaxation of the pyloric smooth muscle, raising the hypothesis that a defect in nitric oxide production could lead to failure of pyloric sphincter relaxation with subsequent hypertrophy. In a pioneering study published twenty years ago, Vanderwinden and colleagues demonstrated absence of nNOS in hypertrophied pyloric muscle tissue in IHPS patients¹⁰. Furthermore, rats and mice with experimentally inhibited or absent nNOS activity are reported to have features of IHPS ^{u> 12}. In IHPS patients, electron microscopy and immunohistochemistry studies suggest that the absence of nNOS may result from deficient innervation of the pyloric circular muscle layer ^13"17.

Several linkage and candidate gene studies have been conducted, but there are no genetic variants with replicated association findings. In search of genetic variants underlying these pathophysiological findings, several association studies have been conducted focusing on NOS1 (the gene encoding nNOS), MLN (the motilin gene), and other functional candidate genes ^{18 24}. However, these studies have been relatively small and results have been conflicting. To identify common sequence variants associated with IHPS as a prelude to a better understanding of its biology and as a tool to identify high-risk individuals, we used a genome-wide association study (GWAS) approach.

The present inventors have studied an ethnically homogeneous population from Denmark with linkage of Electronic Medical Records for diagnosis to a biorepository of blood spots from population based Neonatal Screening in

Denmark to provide samples for DNA extraction. Controls were drawn from the same population and a large independent sample set of cases and controls was available to replicate positive signals.

Summary of the invention

The present invention relates to an in vitro method for diagnosing a genetic predisposition or susceptibility for pyloric stenosis in a mammal, comprising detecting in a sample obtained from said mammal at least one single nucleotide polymorphism (SNP) in a nucleic acid or fragment thereof, wherein said at least one SNP in selected from the group consisting of the SNPs rsl l712066, rs29784, rs573872, rsl l216185, rsl208285, rs2228671, rsl2721025 and SNPs in linkage disequilibrium therewith, whereby at least one allele-specific haplotype is determined.

In an embodiment of the present invention this method further comprises the steps of determining the expression level of at least one of the genes selected from the group consisting of MBNL1, NKX2-5, C3orf79, SGEF, P2RY1, RAP2B, BNIP1, MGC34034, TCF21, AADAC, SUCNR1, SIK3, APOA1, APOC3, APOA4, APOA5, LDLR, DHX36, GPR149, TMEM14E, C5orf41, ATP5VOE1, SNORA74B, PAFAH1B2, SIDT2, TAG UN, PCSK7 and LOC401093, comparing said expression level to a predetermined expression level, and diagnosing said mammal with a genetic predisposition or susceptibility for pyloric stenosis if said at least one allele-specific haplotype is determined and said expression level is significantly different from said predetermined expression level.

Another embodiment of the present invention relates to SNPs in linkage disequilibrium with the SNPs of the present invention. In another embodiment of the present invention is the mammal a human infant selected from the group consisting of a boy and a girl. In yet another embodiment of the present invention is the detection of the SNPs accomplished by sequencing, mini-sequencing, hybridization, restriction fragment analysis, oligonucleotide ligation assay, allele specific PCR. Another aspect of the present invention relates to a diagnostic kit and/or a research kit, comprising at least one combination of probes for detecting at least one of the haplotypes of the present invention and/or probes for determining the expression level the genes of the present invention. Yet another aspect of the present invention relates to the use of cholesterol form preparing a pharmaceutical composition or medical for the treatment of pyloric stenosis in infants.

Figures Figure 1.

Discovery, replication and combined results of the loci associated with pyloric stenosis. One SNP per region was followed up in the replication stage. Eff, effect allele; Alt, alternative allele; CI, confidence interval; Het P, P value for test of heterogeneity using the 12 statistic.

Figure 2.

Regional association plots for three confirmed novel IHPS loci: a) chromosome 3q25.1, b) chromosome 3q25.2, and c) chromosome 5q35.2. SNPs are plotted by chromosomal position (x-axis) against GWAS association with IHPS (-log 10 P value). The strongest signal is labeled in the plot, and other SNPs are color coded to reflect their LD with the top SNP (based on pairwise r² values from the discovery stage data). Estimated recombination rates (from HapMap) are plotted to reflect the local LD structure. Genes are indicated in the lower panel of each plot. The figure was generated using LocusZoom.

Figure 3.

Basic characteristics of the discovery and replication sample, a): The replication cases were born an average of 6 years earlier than discovery cases. This was by design to address the concern that blood spot samples that had been stored for many years might not give as good yield in GWAS genotyping as those stored for fewer years and we therefore prioritized cases born most recently for the discovery stage.

Figure 4.

Discovery, replication and combined results for one additional SNP at each of the six top loci associated with IHPS. These additional SNPs were runner-ups in the discovery analysis and show high correlation to the primary SNPs in the combined discovery and replication sample. Eff, effect allele; Alt, alternative allele; CI, confidence interval; Het P, P value for test of heterogeneity using the I² statistic a): Correlation coefficients (r²) are based on the combined discovery and replication sample apart from rs9389106, where it is based on data from HapMap CEU sample. brs9389106 was not genotyped in the discovery stage; rsl208285 was used as a proxy in the discovery sample.

Figure 5.

Association results for pyloric stenosis stratified by sex and combined. Results are given separately for the discovery data, the replication data, and the combined discovery and replication data. Eff, effect allele; Alt, alternative allele; CI, confidence interval; Het P, P value for test of heterogeneity between results for boys and girls using the I² statistic.

Figure 6.

Quantile-quantile plot of observed versus expected -log 10 P values from the genome-wide scan for IHPS after correction by genomic control (λ=1.06). The expected distribution of -loglO P values under the null hypothesis is shown by the grey line. Figure 7.

Manhattan plot of GWAS for IHPS. The genomewide distribution of -loglO P values after correction by genomic control (λ=1.06) is shown across the chromosomes. Figure 8.

Regional association plots for three possible novel IHPS loci: a) chromosome 6q23.2, b) chromosome llq23.3, and c) chromosome 19pl3.2. SNPs are plotted by chromosomal position (x-axis) against GWAS association with IHPS (-log 10 P value). The strongest signal is labeled in the plot, and other SNPs are color coded to reflect their LD with the top SNP (based on pairwise r² values from the discovery stage data). Estimated recombination rates (from HapMap) are plotted to reflect the local LD structure. Genes are indicated in the lower panel of each plot. The figure was generated using LocusZoom.

Figure 9.

Figure 10.

Regional association plot showing imputed SNP results for the novel IHPS locus on chromosome llq23.3. SNPs are plotted by chromosomal position (x-axis) against GWAS association with IHPS (-loglO P value). The strongest signal is labeled in the plot, and other SNPs are color coded to reflect their LD with the top SNP (based on pairwise r² values from the discovery stage data). Estimated

recombination rates (from HapMap) are plotted to reflect the local LD structure. Genes are indicated in the lower panel of each plot. The figure was generated using LocusZoom.

Figure 11.

Association results for 50 imputed SNPs with P<10^"6 across the llq23.3 locus that also were found to associate with levels of total and/or HDL cholesterol by

Teslovich and colleagues (Nature 2010). The table is sorted by basepair position (NCBI build 37) and shows effect (Eff) and alternative (Alt) allele; effect allele frequency; info value from SNPTEST indicating imputation quality; odds ratio for association with IHPS; P-value (after genomic control, λ=1.05 from discovery scan); squared Pearson correlation coefficient (r2) of imputed SNP allele dosage to allele dosage for the top SNP at the locus (rsl2721025); lipid trait; effect allele for association with lipid trait; effect estimate and P value for the lipid trait association.

Detailed description in the invention

The present invention discloses polymorphic variants and haplotypes that have been found to be associated with pyloric stenosis. Particular alleles at polymorphic markers to be associated with pyloric stenosis. Such markers and haplotypes are useful for diagnostic purposes.

The present invention also discloses genes that are relevant for the development and diagnosis of pyloric stenosis. These genes are either over expressed or under expressed in comparison to a normal phenotype.

Further applications of the present invention includes methods kits for use in the methods of the invention an methods of treatment of pyloric stenosis.

A first aspect of the present invention relates to an in vitro method for diagnosing a genetic predisposition or susceptibility for pyloric stenosis in a mammal, comprising detecting in a sample obtained from said mammal at least one single nucleotide polymorphism (SNP) in a nucleic acid or fragment thereof, wherein said at least one SNP in selected from the group consisting of the SNPs rsl 1712066, rs29784, rs573872, rsl l216185, rsl208285, rs2228671, rsl2721025 and SNPs in linkage disequilibrium therewith, whereby at least one allele-specific haplotype is determined.

This allele-specific haplotype can be a direct indicator of a genetic predisposition or susceptibility for pyloric stenosis or it can suggest that an elevated risk which can, optionally, be further supported in combination with the expression profiling of specific genes that are relevant for the development and diagnosis of pyloric stenosis. Thus in an embodiment of the present invention comprises the above method the further steps of determining the expression level of at least one of the genes selected from the group consisting of MBNL1, NKX2-5, C3orf79, SGEF, P2RY1, RAP2B, BNIP1, MGC34034, TCF21, AADAC, SUCNR1, SIK3, APOA1, APOC3, APOA4, APOA5 and LDLR, DHX36, GPR149, TMEM14E, C5orf41, ATP6VOE1, SNORA74B, PAFAH1B2, SIDT2, TAGLN, PCSK7 and LOC401093,comparing said expression level to a predetermined expression level, and diagnosing said mammal with a genetic predisposition or susceptibility for pyloric stenosis if said at least one allele-specific haplotype is determined and said expression level is significantly different from said predetermined expression level.

In one embodiment of the present invention is at least one, such as at least two, such as at least three, such as at least four, such as at least five, such as six SNP(s) detected in the above method including, optionally, at least one gene, such as 2 genes, such as 3 genes, such as 4 genes, such as 5 genes, such as 6 genes, such as 7 genes, such as 9 genes, such as 10 genes, such as 12 genes, such as 15 genes, such as 20 genes, such as 25 genes or variants hereof selected from the group consisting of MBNL1, NKX2-5, C3orf79, SGEF, P2RY1, RAP2B, BNIP1, MGC34034, TCF21, AADAC, SUCNR1, SIK3, APOA1, APOC3, APOA4, APOA5 and LDLR, DHX36, GPR149, TMEM14E, C5orf41, ATP6VOE1, SNORA74B, PAFAH1B2, SIDT2, TAGLN, PCSK7 and LOC401093.

Definitions

The following terms shall, in the present context, have the meaning as indicated:

Pyloric stenosis, pyloric stenosis in infants, or infantile hypertrophic pyloric stenosis (IHPS) is a condition that causes severe vomiting in the first few months of life. There is narrowing (stenosis) of the opening from the stomach to the intestines, due to enlargement (hypertrophy) of the muscle surrounding this opening (the pylorus, meaning "gate"), which spasms when the stomach empties.

It is uncertain whether there is a real congenital narrowing or whether there is a functional hypertrophy of the muscle which develops in the first few weeks of life. Pyloric stenosis also occurs in adults where the cause is usually a narrowed pylorus due to scarring from chronic peptic ulceration. This is a different condition from the infantile form. A "polymorphic marker", sometimes referred to as a "marker", "single nucleotide polymorphism" or "SNP" as described herein, refers to a genomic polymorphic site. Each polymorphic marker has at least two sequence variations characteristic of particular alleles at the polymorphic site. An "allele" refers to the nucleotide sequence of a given locus (position) on a chromosome. A polymorphic marker allele thus refers to the composition (i.e., sequence) of the marker on a chromosome.

Genomic DNA from an individual contains two alleles for any given polymorphic marker, representative of each copy of the marker on each chromosome.

Sequence codes for nucleotides used herein are: A = 1, C = 2, G = 3, T = 4.

A "Single Nucleotide Polymorphism" or "SNP" is a DNA sequence variation occurring when a single nucleotide at a specific location in the genome differs between members of a species or between paired chromosomes in an individual.

Most SNP polymorphisms have two alleles. Each individual is in this instance either homozygous for one allele of the polymorphism (i.e. both chromosomal copies of the individual have the same nucleotide at the SNP location), or the individual is heterozygous (i.e. the two sister chromosomes of the individual contain different nucleotides).

The SNP nomenclature as reported herein refers to the official Reference SNP (rs) ID identification tag as assigned to each unique SNP by the National Center for Biotechnological Information (NCBI).

Sequence conucleotide ambiguity as described herein is as proposed by IUPAC- IUB. These codes are compatible with the codes used by the EMBL, GenBank, and PIR databases. A nucleotide position at which more than one sequence is possible in a population (either a natural population or a synthetic population, e.g., a library of synthetic molecules) is referred to herein as a "polymorphic site". A "variant", as described herein, refers to a segment of DNA that differs from the reference DNA. A "marker" or a "polymorphic marker", as defined herein, is a variant. Alleles that differ from the reference are referred to as "variant" alleles. -

A "haplotype," as described herein, refers to a segment of genomic DNA within one strand of DNA that is characterized by a specific combination of alleles arranged along the segment. For diploid organisms such as humans, a haplotype comprises one member of the pair of alleles for each polymorphic marker or locus. In a certain embodiment, the haplotype can comprise two or more alleles, three or more alleles, four or more alleles, or five or more alleles.

Alternatively, the markers and/or haplotypes of the invention are characteristic of decreased susceptibility (i.e., decreased risk) of pyloric stenosis, as characterized by a relative risk of less than one, or an odds ratio of less than one. Haplotypes are described herein in the context of the marker name and the allele of the marker in that haplotype, e.g., "T rs4415084" refers to the T allele of marker rs4415084 being in the haplotype, and this nomenclature is equivalent to

"rs4415084 allele T" and "T-rs4415084". Furthermore, allelic codes in haplotypes are as for individual markers, i.e. I = A, 2 = C, 3 = G and 4 = T.

The MBNL1, NKX2-5, C3orf79, SGEF, P2RY1, RAP2B, BNIP1, MGC34034, TCF21, AADAC, SUCNRl, SIK3, APOAl, APOC3, AP0A4, APOA5, LDLR, DHX36, GPR149, TMEM14E, C5orf41, ATP6VOE1, SNORA74B, PAFAH1B2, SIDT2, TAGLN, PCSK7 and LOC401093 genes are also known as Muscleblind-like 1, Homeobox protein Nkx-2.5, chromosome 3 open reading frame 79, Src homology 3 domain- containing Guanine nucleotide Exchange Factor, P2Y purinoceptor 1, Ras-related protein Rap-2b, BCL2/adenovirus EIB 19kDa interacting protein 1, MGC34034, Transcription factor 21, Arylacetamide deacetylase, Succinate receptor 1,

Serine/threonine-protein kinase SIK3, Apolipoprotein A-I, Apolipoprotein C-III, Apolipoprotein A- IV, Apolipoprotein A-V, low density lipoprotein receptor, DEAH box protein 36, G protein-coupled receptor 149, transmembrane protein 14E, Chromosome 5 open reading frame 41, ATP6VOE1, Small nucleolar RNA H/ACA box 74B, platelet-activating factor acetylhydrolase lb catalytic subunit 2 (30kDa), SID1 transmembrane family member 2, Transgelin, Proprotein convertase subtilisin/kexin type 7, and LOC401093, respectively. The term "susceptibility", as described herein, refers to the proneness of an individual towards the development of a certain state (e.g., a certain trait, phenotype or disease, e.g., pyloric stenosis), or towards being less able to resist a particular state than the average individual. The term encompasses both increased susceptibility and decreased susceptibility.

Thus, particular alleles at polymorphic markers and/or haplotypes of the invention as described herein may be characteristic of increased susceptibility (i.e., increased risk) of pyloric stenosis, as characterized by a relative risk (RR) or odds ratio (OR) of greater than one for the particular allele or haplotype. Alternatively, the markers and/or haplotypes of the invention are characteristic of decreased susceptibility (i.e., decreased risk) of pyloric stenosis, as characterized by a relative risk of less than one.

The term "and/or" shall in the present context be understood to indicate that either or both of the items connected by it are involved. In other words, the term herein shall be taken to mean "one or the other or both".

A "nucleic acid sample" is a sample obtained from an individual that contains nucleic acid (DNA or RNA). In certain embodiments, i.e. the detection of specific polymorphic markers and/or haplotypes, the nucleic acid sample comprises genomic DNA. Such a nucleic acid sample can be obtained from any source that contains genomic DNA, including as a blood sample, sample of amniotic fluid, sample of cerebrospinal fluid, or tissue sample from skin, muscle, buccal or conjunctival mucosa, placenta, gastrointestinal tract or other organs.

Assessment for markers and haplotypes

The genomic sequence within populations is not identical when individuals are compared. Rather, the genome exhibits sequence variability between individuals at many locations in the genome. Such variations in sequence are commonly referred to as polymorphisms, and there are many such sites within each genome. For example, the human genome exhibits sequence variations which occur on average every 500 base pairs. The most common sequence variant consists of base variations at a single base position in the genome, and such sequence variants, or polymorphisms, are commonly called Single Nucleotide Polymorphisms ("SNPs").

These SNPs are believed to have arisen by a single mutational event, and therefore there are usually two possible alleles possible at each SNP site; the original allele and the mutated (alternate) allele. Due to natural genetic drift and possibly also selective pressure, the original mutation has resulted in a

polymorphism characterized by a particular frequency of its alleles in any given population.

In general terms, polymorphisms can comprise any number of specific alleles. Thus in one embodiment of the invention, the polymorphism is characterized by the presence of two or more alleles in any given population. In another embodiment, the polymorphism is characterized by the presence of three or more alleles.

In other embodiments, the polymorphism is characterized by four or more alleles, five or more alleles, six or more alleles, seven or more alleles, nine or more alleles, or ten or more alleles.

All such polymorphisms can be utilized in the methods and kits of the present invention, and are thus within the scope of the invention.

In some instances, reference is made to different alleles at a polymorphic site without choosing a reference allele. Alternatively, a reference sequence can be referred to for a particular polymorphic site. The reference allele is sometimes referred to as the "wild-type" allele and it usually is chosen as either the first sequenced allele or as the allele from a "non-affected" individual (e.g., an individual that does not display a trait or disease phenotype). Alleles for SNP markers as referred to herein refer to the bases A, C, G or T as they occur at the polymorphic site in the SNP assay employed. The allele codes for SNPs used herein are as follows: 1= A, 2=C, 3=G, 4=T. The person skilled in the art will however realize that by assaying or reading the opposite DNA strand, the complementary allele can in each case be measured.

Thus, for a polymorphic site (polymorphic marker) characterized by an A/G polymorphism, the assay employed may be designed to specifically detect the presence of one or both of the two bases possible, i.e. A and G. Alternatively, by designing an assay that is designed to detect the opposite strand on the DNA template, the presence of the complementary bases T and C can be measured.

Quantitatively (for example, in terms of relative risk), identical results would be obtained from measurement of either DNA strand (+ strand or - strand).

Typically, a reference sequence is referred to for a particular sequence. Alleles that differ from the reference are sometimes referred to as "variant" alleles. A variant sequence, as used herein, refers to a sequence that differs from the reference sequence but is otherwise substantially similar. Alleles at the

polymorphic genetic markers described herein are variants. Variants can include changes that affect a polypeptide.

A haplotype refers to a segment of DNA that is characterized by a specific combination of alleles arranged along the segment. For diploid organisms such as humans, a haplotype comprises one member of the pair of alleles for each polymorphic marker or locus.

In a certain embodiment, the haplotype can comprise two or more alleles, three or more alleles, four or more alleles, or five or more alleles, each allele

corresponding to a specific polymorphic marker along the segment. Haplotypes can comprise a combination of various polymorphic markers, e.g., SNPs and microsatellites, having particular alleles at the polymorphic sites. The haplotypes thus comprise a combination of alleles at various genetic markers. Detecting specific polymorphic markers and/or haplotypes can be accomplished by methods known in the art for detecting sequences at polymorphic sites. For example, standard techniques for genotyping for the presence of SNPs and/or microsatellite markers can be used, such as fluorescence-based techniques, utilizing PCR, LCR, Nested PCR and other techniques for nucleic acid amplification.

Specific commercial methodologies available for SNP genotyping include, but are not limited to, TaqMan genotyping assays and SNPIex platforms (Applied

Biosystems), gel electrophoresis (Applied Biosystems), mass spectrometry (e.g., MassARRAY system from Sequenom), minisequencing methods, real-time PCR, Bio-Plex system (BioRad), CEQ and SNPstream systems (Beckman), array hybridization technology (e.g., Affymetrix GeneChip; Perlegen), BeadArray Technologies (e.g., Illumina GoldenGate and Infinium assays), array tag technology (e.g., Parallele), and endonuclease-based fluorescence hybridization technology (Invader; Third Wave).

In certain methods described herein, an individual who is at an increased susceptibility (i.e., increased risk) for pyloric stenosis, is an individual in whom at least one specific allele at one or more polymorphic marker or haplotype conferring increased susceptibility for pyloric stenosis is identified (i.e., at-risk marker alleles or haplotypes).

In one aspect, the at-risk marker or haplotype is one that confers a significant increased risk (or susceptibility) of pyloric stenosis.

In one embodiment, significance associated with a marker or haplotype is measured by a relative risk (RR). In another embodiment, significance associated with a marker or haplotye is measured by an odds ratio (OR). In a further embodiment, the significance is measured by a percentage.

In one embodiment, a significant increased risk is measured as a risk (relative risk and/or odds ratio) of at least 1.10, including but not limited to: at least 1.11, at least 1.12, at least 1.13, at least 1.14, at least 1.15, at least 1.16, at least 1.17, at least 1.18, at least 1.19, at least 1.20, at least 1.21, at least 1.22, at least 1.23, at least 1.24, at least 1.25, at least 1.30, at least 1.35, at least 1.40, at least 1.50, at least 1.60, at least 1.70, 1.80, at least 1.90, at least 2.0, at least 2.5, at least 3.0, at least 4.0, and at least 5.0. In a particular embodiment, a risk (relative risk and/or odds ratio) of at least 1.15 is significant. In another particular 5 embodiment, a risk of at least 1.17 is significant.

In yet another embodiment, a risk of at least 1.20 is significant. In a further embodiment, a relative risk of at least about 1.25 is significant. In another furthe embodiment, a significant increase in risk is at least about 1.30 is significant. In

10 yet another further embodiment, a significant increase in risk is at least about 1.40 is significant. In yet another further embodiment, a significant increase in risk is at least about 1.50 is significant. In yet another further embodiment, a significant increase in risk is at least about 1.60 is significant. In yet another further embodiment,^ significant increase in risk is at least about 1.70 is

15 significant. In yet another further embodiment, a significant increase in risk is at least about 1.80 is significant.

However, other cutoffs are also contemplated, e.g. at least 1.16, 1.18, 1.19, 1.21, 1.22, and so on, and such cutoffs are also within scope of the present invention.

20

In other embodiments, a significant increase in risk is at least about 5%, including but not limited to about 10 %, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, 25 and about 100%. In one particular embodiment, a significant increase in risk is at least 15%.

In other embodiments, a significant increase in risk is at least 17%, at least 20%, at least 22%, at least 24%, at least 25%, at least 30%, at least 32% and at least 30 35%. Other cutoffs or ranges as deemed suitable by the person skilled in the art to characterize the invention are however also contemplated, and those are also within scope of the present invention.

In certain embodiments, a significant increase in risk is characterized by a p- 35 value, such as a p-value of less than 0.05, less than 0.01, less than 1 x 10^"3 (0.001), less than 1 x 10^"4 (0.0001), less than 1 x 10^~4 (0.00001), less than 1 x 10^"5 (0.000001), less than 1 x 10^"6 (0.0000001), less than 1 x 10^"7 (0.00000001), less than 1 x 10^"8 (0.000000001), less than 1 x 10^"10 (0.00000000001), less than 1 x 10^"12 (0.0000000000001), less than 1 x 10^"14 (0.000000000000001), less than 1 x 10^"16 (0.00000000000000001), or less than 1 x 10^"18

(0.0000000000000000001).

An at-risk polymorphic marker or haplotype of the present invention is one where at least one allele of at least one marker or at least one haplotype is more frequently present in an individual at risk for the disease or trait (affected), or diagnosed with the disease or trait, compared to the frequency of its presence in a comparison group (control), such that the presence of the allele or haplotype is indicative of susceptibility to the disease or trait (e.g., pyloric stenosis). The control group may in one embodiment be a population sample, i.e. a random sample from the general population. In another embodiment, the control group is represented by a group of individuals who are disease-free, i.e. individuals who have not been diagnosed with pyloric stenosis. Such disease-free control may in one embodiment be characterized by the absence of one or more specific disease- associated symptoms.

As an example of a simple test for association would be a Fisher-exact test on a two by two table. Given a group of cases and a group of controls, the two by two table is constructed with one row for the cases and one for the controls. In the first column, the number of chromosomes that has the specific allele (or haplotype) is counted. In the second column the number of chromosomes that does not have the allele (or haplotype) is counted.

Other statistical tests of association known to the skilled person are also contemplated and are also within scope of the invention.

In other embodiments of the invention, an individual who is at a decreased susceptibility (i.e., at a decreased risk) for a disease or trait is an individual in whom at least one specific allele at one or more polymorphic marker or haplotype conferring decreased susceptibility for the disease or trait is identified. The marker alleles and/or haplotypes conferring decreased risk are also said to be protective. In one aspect, the protective marker or hapiotype is one that confers a significant decreased risk (or susceptibility) of the disease or trait.

In one embodiment, significant decreased risk is measured as a relative risk of less than 0.90, including but not limited to less than 0.85, less than 0.80, less than 0.75, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2 and less than 0.1. In one particular embodiment, significant decreased risk is less than 0.90.

In another embodiment, significant decreased risk is less than 0.85. In yet another embodiment, significant decreased risk is less than 0.80. In another embodiment, the decrease in risk (or susceptibility) is at least 10%, including but not limited to at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% and at least 98%. In one particular embodiment, a significant decrease in risk is at least about 15%.

In another embodiment, a significant decrease in risk at least about 20%. In another embodiment, the decrease in risk is at least about 25%. Other cutoffs or ranges as deemed suitable by the person skilled in the art to characterize the invention are however also contemplated, and those are also within scope of the present invention.

The person skilled in the art will appreciate that for polymorphic markers with two alleles present in the population being studied (such as SNPs), and wherein one allele is found in increased frequency in a group of individuals with a trait or disease in the population, compared with controls, the other allele of the marker will be found in decreased frequency in the group of individuals with the trait or disease, compared with controls. In such a case, one allele of the marker (the one found in increased frequency in individuals with the trait or disease) will be the at-risk allele, while the other allele will be a protective allele. A genetic variant associated with a disease or a trait (e.g. pyloric stenosis) can be used alone to predict the risk of the disease for a given genotype. For a biallelic marker, such as a SNP, there are 3 possible genotypes: homozygote for the at risk variant, heterozygote, and non carrier of the at risk variant. Risk associated with variants at multiple loci can be used to estimate overall risk. For multiple SNP variants, there are k possible genotypes k = 3" x 2"; where n is the number autosomal loci and p the number of gonosomal (sex chromosomal) loci. Overall risk assessment calculations usually; assume that the relative risks of different genetic variants multiply, i.e. the overall risk (e.g., RR or OR) associated with a particular genotype combination is the product of the risk values for the genotype at each locus.

If the risk presented is the relative risk for a person, or a specific genotype for a person, compared to a reference population with matched gender and ethnicity, then the combined risk - is the product of the locus specific risk values - and which also corresponds to an overall risk estimate compared with the population.

If the risk for a person is based on a comparison to non-carriers of the at risk allele, then the combined risk corresponds to an estimate that compares the person with a given combination of genotypes at all loci to a group of individuals who do not carry risk variants at any of those loci.

The group of non-carriers of any at risk variant has the lowest estimated risk and has a combined risk, compared with itself {i.e., non-carriers) of 1.0, but has an overall risk, compare with the population, of less than 1.0. It should be noted that the group of non-carriers can potentially be very small, especially for large number of loci, and in that case, its relevance is correspondingly small. Expression profiling of pyloric stenosis associated genes

One embodiment of the present invention relates to determining the expression level of at least one of the genes selected from the group consisting of MBNL1, NKX2-5, C3orf79, SGEF, P2RY1, RAP2B, BNIP1, MGC34034, TCF21, AADAC, SIJCNRl, SIK3, APOA1, APOC3, APOA4, APOA5 and LDLR, DHX36, GPR149, TMEM14E, C5orf41, ATP6VOE1, SNORA74B, PAFAH1B2, SIDT2, TAGLN, PCSK7 and LOC401093, comparing said expression level to a predetermined expression level, and diagnosing said mammal with a genetic predisposition or susceptibility for pyloric stenosis if said at least one allele-specific haplotype is determined and said expression level is significantly different from said predetermined expression level. The predetermined expression level can be from a normal population wherein no pyloric stenosis has been diagnosed, similar to the populations described for SNPs in the present application.

A comparison of an expression level to a predetermined expression level such as the expression level from a normal population can be done using certain specificities and sensitivities using statistics.

Expression level

The expression level of a given genetic element as used herein refers to the absolute or relative amount of RNA corresponding to this genetic element in a given sample. Expressed genes include genes that are transcribed into mRNA and then translated into protein, as well as genes that are transcribed into mRNA, or other types of RNA such as, tRNA, rRNA or other non-coding RNAs, that are not translated into protein. RNA expression is a highly specific process which can be monitored by detecting the absolute or relative RNA levels.

Thus, the expression level refers to the amount of RNA in a sample. The expression level is usually detected using microarrays, northern blotting, RT-PCR, SAGE, RNA-seq, or similar RNA detection methods. When expression levels of a specific RNA in a test sample is compared to a reference sample they can either be different or equal. However, using today's detection techniques is an exact definition of different or equal result can be difficult because of noise and variations in obtained expression levels from different samples. Hence, the usual method for evaluating whether two or more expression levels are different or equal involves statistics.

Statistics enables evaluation of significantly different expression levels and significantly equal expressions levels. Statistical methods involve applying a function/statistical algorithm to a set of data. Statistical theory defines a statistic as a function of a sample where the function itself is independent of the sample's distribution: the term is used both for the function and for the value of the function on a given sample. Commonly used statistical tests or methods applied to a data set include t-test, f-test or even more advanced test and methods of comparing data. Using such a test or methods enables a conclusion of whether two or more samples are significantly different or significantly equal.

Sensitivity

As used herein the sensitivity refers to the measures of the proportion of actual positives which are correctly identified as such - in analogy with a diagnostic test, i.e. the percentage of sick people who are identified as having the condition.

Usually the sensitivity of a test can be described as the proportion of true positives of the total number with the target disorder. All patients with the target disorder are the sum of (detected) true positives (TP) and (undetected) false negatives (FN).

Specificity

As used herein the specificity refers to measures of the proportion of negatives which are correctly identified - i.e. the percentage of well people who are identified as not having the condition. The ideal diagnostic test is a test that has 100 % specificity, i.e. only detects diseased individuals and therefore no false positive results, and 100 % sensitivity, i.e. detects all diseased individuals and therefore no false negative results.

For any test, there is usually a trade-off between each measure. For example in a manufacturing setting in which one is testing for faults, one may be willing to risk discarding functioning components (low specificity), in order to increase the chance of identifying nearly all faulty components (high sensitivity). This trade-off can be represented graphically using a (receiver operator characteristic) ROC curve.

Selecting a sensitivity and specificity it is possible to obtain the optimal outcome in a detection method. In determining the discriminating value distinguishing subjects or individuals having or developing e.g. pyloric stenosis, the person skilled in the art has to predetermine the level of specificity. The ideal diagnostic test is a test that has 100% specificity, i.e. only detects diseased individuals and therefore no false positive results, and 100% sensitivity, i.e. detects all diseased individuals and therefore no false negative results. However, due to biological diversity no method can be expected to have 100% sensitive without including a substantial number of false negative results.

The chosen specificity determines the percentage of false positive cases that can be accepted in a given study/population and by a given institution. By decreasing specificity an increase in sensitivity is achieved. One example is a specificity of 95% which will result in a 5% rate of false positive cases. With a given prevalence of 1% of e.g. pyloric stenosis in a screening population, a 95% specificity means that 5 individuals out of 100 will undergo further physical examination in order to detect one pyloric stenosis case if the sensitivity of the test is 100%.

The cut-off level could be established using a number of methods, including: " percentiles, mean plus or minus standard deviation(s); multiples of median value; patient specific risk or other methods known to those who are skilled in the art. Linkage Disequilibrium

The natural phenomenon of recombination, which occurs on average once for each chromosomal pair during each meiotic event, represents one way in which nature provides variations in sequence (and biological function by consequence).

It has been discovered that recombination does not occur randomly in the genome; rather, there are large variations in the frequency of recombination rates, resulting in small regions of high recombination frequency (also called recombination hotspots) and larger regions of low recombination frequency, which are commonly referred to as Linkage Disequilibrium (LD) blocks.

Linkage Disequilibrium (LD) refers to a non-random assortment of two genetic elements. For example, if a particular genetic element (e.g., an allele of a polymorphic marker, or a haplotype) occurs in a population at a frequency of 0.50 (50%) and another element occurs at a frequency of 0.50 (50%), then the predicted occurrence of a person's having both elements is 0.25 (25%), assuming a random distribution of the elements.

However, if it is discovered that the two elements occur together at a frequency higher than 0.25, then the elements are said to be in linkage disequilibrium, since they tend to be inherited together at a higher rate than what their independent frequencies of occurrence (e.g., allele or haplotype frequencies) would predict.

Roughly speaking, LD is generally correlated with the frequency of recombination events between the two elements. Allele or haplotype frequencies can be determined in a population by genotyping individuals in a population and determining the frequency of the occurrence of each allele or haplotype in the population. For populations of diploids, e.g., human populations, individuals will typically have two alleles for each genetic element (e.g., a marker, haplotype or gene).

Many different measures have been proposed for assessing the strength of linkage disequilibrium. Most capture the strength of association between pairs of biallelic sites. Two important pairwise measures of LD are r² (sometimes denoted DA2) and |D'| . Both measures range from 0 (no disequilibrium) to 1 ('complete' disequilibrium), but their interpretation is slightly different.

|D'| is defined in such a way that it is equal to 1 if just two or three of the possible haplotypes are present, and it is < 1 if all four possible haplotypes are present.

Therefore, a value of |D'| that is <1 indicates that historical recombination may have occurred between two sites (recurrent mutation can also cause |D'| to be < 1, but for single nucleotide polymorphisms (SNPs) this is usually regarded as being less likely than recombination).

The measure r² represents the statistical correlation between two sites, and takes the value of 1 if only two haplotypes are present.

The r² measure is arguably the most relevant measure for association mapping, because there is a simple inverse relationship between r2 and the sample size required to detect association between susceptibility loci and particular SNPs. These measures are defined for pairs of sites, but for some applications a determination of how strong LD is across an entire region that contains many polymorphic sites might be desirable (e.g., testing whether the strength of LD differs significantly among loci or across populations, or whether there is more or less LD in a region than predicted under a particular model).

Measuring LD across a region is not straightforward, but one approach is to use the measure r, which was developed in population genetics. Roughly speaking, r measures how much recombination would be required under a particular population model to generate the LD that is seen in the data.

This type of method can potentially also provide a statistically rigorous approach to the problem of determining whether LD data provide evidence for the presence of recombination hotspots. For the methods described herein, a significant r2 value between genetic segments (such as SNP markers) can be at least 0.1 such as at least 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99 or 1.0.

In one preferred embodiment, the significant r² value can be at least 0.2.

Alternatively, linkage disequilibrium as described herein, refers to linkage disequilibrium characterized by values of |D'| of at least 0.2, such as 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.85, 0.9, 0.95, 0.96, 0.97, 0.98, 0.99.

Thus, linkage disequilibrium represents a correlation between alleles of distinct markers. It is measured by correlation coefficient or | D'| (r² up to 1.0 and | D'| up to 1.0).

Linkage disequilibrium can be determined in a single human population, as defined herein, or it can be determined in a collection of samples comprising individuals from more than one human population.

In one embodiment of the invention, LD is determined in a sample from one or more of the HapMap populations (Caucasian, West African, Japanese, Chinese). In one such embodiment, LD is determined in the CEU population of the HapMap samples.iln another embodiment, LD is determined in the Y I population.

In another embodiment, LD is determined in a European population. In yet another embodiment, LD is determined in the Danish population.

If all polymorphisms in the genome were identical at the population level, then every single one of them would need to be investigated in association studies.

However, due to linkage disequilibrium between polymorphisms, tightly linked polymorphisms are strongly correlated, which reduces the number of

polymorphisms that need to be investigated in an association study to observe a significant association.

Another consequence of LD is that many polymorphisms may give an association signal due to the fact that these polymorphisms are strongly correlated. Genomic LD maps have been generated across the genome, and such LD maps have been proposed to serve as framework for mapping disease-genes. It is now established that many portions of the human genome can be broken into series of discrete haplotype blocks containing a few common haplotypes; for these blocks, linkage disequilibrium data provides little evidence indicating

recombination. There are two main methods for defining these haplotype blocks: blocks can be defined as regions of DNA that have limited haplotype diversity, or as regions between transition zones having extensive historical recombination, identified using linkage disequilibrium. More recently, a fine-scale map of recombination rates and corresponding hotspots across the human genome has been generated. The map reveals the enormous variation in recombination across the genome, with recombination rates as high as 10-60 cM/Mb in hotspots, while closer to 0 in intervening regions, which thus represent regions of limited haplotype diversity and high LD. The map can therefore be used to define haplotype blocks/LD blocks as regions flanked by recombination hotspots.

As used herein, the terms "haplotype block" or "LD block" includes blocks defined by any of the above described characteristics, or other alternative methods used by the person skilled in the art to define such regions.

It has thus become apparent that for any given observed association to a polymorphic marker in the genome, it is likely that additional markers in the genome also show association. This is a natural consequence of the uneven distribution of LD across the genome, as observed by the large variation in recombination rates.

The markers used to detect association thus in a sense represent "tags" for a genomic region (i.e., a haplotype block or LD block) that is associating with a given disease or trait. One or more causative (functional) variants or mutations may reside within the region found to be associating to the disease or trait.

The functional variant may be another SNP, a tandem repeat polymorphism (such as a minisatellite or a microsatellite), a transposable element, or a copy number variation, such as an inversion, deletion or insertion. Such variants in LD with the variants described herein may confer a higher relative risk (RR) or odds ratio (OR) than observed for the tagging markers used to detect the association. The present invention thus refers to the markers used for detecting association to the disease, as described herein, as well as markers in linkage disequilibrium with the markers. Thus, in certain embodiments of the invention, markers that are in LD with the markers and/or haplotypes of the invention, as described herein, may be used as surrogate markers.

The surrogate markers have in one embodiment relative risk (RR) and/or odds ratio (OR) values smaller than for the markers or haplotypes initially found to be associating with the disease, as described herein. In other embodiments, the surrogate markers have RR or OR values greater than those initially determined for the markers initially found to be associating with the disease, as described herein.

An example of such an embodiment would be a rare, or relatively rare (< 10% allelic population frequency) variant in LD with a more common variant (> 10% population frequency) initially found to be associating with the disease, such as the variants described herein.

Identifying and using such markers for detecting the association discovered by the inventors as described herein can be performed by routine methods well known to the person skilled in the art, and are therefore within the scope of the present invention.

One embodiment of the present invention relates to SNPs in linkage disequilibrium as set forth in the tables. These SNPs in linkage disequilibrium with the SNPs of the present invention are merely examples of SNPs that are in linkage disequilibrium. Many more exist and can be easily identified using the knowledge of linkage disequilibrium and SNPs mentioned within this disclosure along with general knowledge within the boundaries of the person skilled in the art.

Determination of haplotype frequency

The frequencies of hapiotypes in patient and control groups can be estimated using the expectation-maximization algorithm.

An implementation of this algorithm that can handle missing genotypes and uncertainty with the phase can be used. Under the null hypothesis, the patients and the controls are assumed to have identical frequencies.

Using a likelihood approach, an alternative hypothesis is tested, where a candidate at-risk-haplotype, which can include the markers described herein, is allowed to have a higher frequency in patients than controls, while the ratios of the frequencies of other hapiotypes are assumed to be the same in both groups. Likelihoods are maximized separately under both hypotheses and a corresponding 1-df likelihood ratio statistic is used to evaluate the statistical significance. To look for at-risk and protective markers and hapiotypes within a susceptibility region, for example, within an LD block region, association of all possible combinations of genotyped markers within the region is studied.

The combined patient and control groups can be randomly divided into two sets, equal in size to the original group of patients and controls. The marker and haplotype analysis is then repeated and the most significant p-value registered is determined. This randomization scheme can be repeated, for example, over 100 or 1000 times to construct an empirical distribution of p-values. In a preferred embodiment, a p- value of <0,05 is indicative of an significant marker and/or haplotype association. Risk assessment and Diagnostics

The Risk presented is usually the relative risk for a person, or a specific genotype of a person, compared to the population with matched gender and ethnicity. Risks of two individuals of the same gender and ethnicity could be compared in a simple manner.

For example, if, compared to the population, the first individual has relative risk 1.5 and the second has relative risk 0.5, then the risk of the first individual compared to the second individual is 1.5/0.5 = 3.

As described herein, certain polymorphic markers and haplotypes comprising such markers are found to be useful for risk assessment of pyloris stenosis. Risk assessment can involve the use of the markers for diagnosing a susceptibility to pyloric stenosis. Particular alleles of polymorphic markers are found more frequently in individuals with pyloric stenosis, than in individuals without diagnosis of pylori stenosis.

Therefore, these marker alleles have predictive value for detecting pyloric stenosis, or a susceptibility to pyloric stenosis, in an individual. Tagging markers within haplotype blocks or LD blocks comprising at-risk markers, such as the markers of the present invention, can be used as surrogates for other markers and/or haplotypes within the haplotype block or LD block.

Markers with values of r² equal to 1 are perfect surrogates for the at-risk variants, i.e. genotypes for one marker perfectly predicts genotypes for the other. Markers with smaller values of r² than 1 can also be surrogates for the at-risk variant, or alternatively represent variants with relative risk values as high or possibly even higher than the at-risk variant. The at-risk variant identified may not be the functional variant itself, but is in this instance in linkage disequilibrium with the true functional variant. The functional variant may for example be a tandem repeat, such as a minisatellite or a microsatellite, a transposable element (e.g., an AIu element), or a structural alteration, such as a deletion, insertion or inversion (sometimes also called copy number variations, or CNVs).

The present invention encompasses the assessment of such surrogate markers for the markers as disclosed herein. Such markers are annotated, mapped and listed in public databases, as well known to the skilled person, or can alternatively be readily identified by sequencing the region or a part of the region identified by the markers of the present invention in a group of individuals, and identify

polymorphisms in the resulting group of sequences. As a consequence, the person skilled in the art can readily and without undue experimentation genotype surrogate markers in linkage disequilibrium with the markers and/or haplotypes as described herein.

The tagging or surrogate markers in LD with the at-risk variants detected, also have predictive value for detecting association to pyloric stenosis, or a

susceptibility to pyloric stenosis, in an individual.

These tagging or surrogate markers that are in LD with the markers of the present invention can also include other markers that distinguish among haplotypes, as these similarly have predictive value for detecting susceptibility to pyloric stenosis.

The present invention can in certain embodiments be practiced by assessing a sample comprising genomic DNA from an individual for the presence of variants described herein to be associated with pyloric stenosis.

Such assessment includes steps of detecting the presence or absence of at least one allele of at least one polymorphic marker, using methods well known to the skilled person and further described herein, and based on the outcome of such assessment, determine whether the individual from whom the sample is derived is at increased or decreased risk (increased or decreased susceptibility) of pyloric stenosis.

Alternatively, the invention can be practiced utilizing a dataset comprising information about the genotype status of at least one polymorphic marker described herein to be associated with pyloric stenosis (or markers in linkage disequilibrium with at least one marker shown herein to be associated with pyloric stenosis). In other words, a dataset containing information about such genetic status, for example in the form of genotype counts at a certain polymorphic marker, or a plurality of markers (e.g., an indication of the presence or absence of certain at- risk alleles), or actual genotypes for one or more markers, can be queried for the presence or absence of certain at-risk alleles at certain polymorphic markers shown by the present inventors to be associated with pyloric stenosis.

A positive result for a variant (e.g., marker allele) associated with increased risk of pyloric stenosis, as shown herein, is indicative of the individual from which the dataset is derived is at increased susceptibility (increased risk) of pyloric stenosis.

In certain embodiments of the invention, a polymorphic marker is correlated to pyloric stenosis by referencing genotype data for the polymorphic marker to a look-up table that comprises correlations between at least one allele of the polymorphism and pyloric stenosis.

In some embodiments, the table comprises a correlation for one polymorhpism. In other embodiments, the table comprises a correlation for a plurality of

polymorphisms. In both scenarios, by referencing to a look-up table that gives an indication of a correlation between a marker and pyloric stenosis, a risk for pyloric stenosis, or a susceptibility to pyloric stenosis, can be identified in the individual from whom the sample is derived. In some embodiments, the correlation is reported as a statistical measure. The statistical measure may be reported as a risk measure, such as a relative risk ( ), an absolute risk (AR) or an odds ratio (OR). Other embodiments include the use of the variants of the present invention in combination with other variants known to be useful for diagnosing a susceptibility -to pyloric stenosis.

Results for any two or more markers can be combined in such analysis, such as results for three markers, four markers, five markers, six markers, seven markers, eight markers, nine markers, or ten or more markers.

Thus, one embodiment of the present invention relates to a at least one SNP is one SNP, such as 2 SNPs, such as 3 SNPs, such as 4 SNPs, such as 5 SNPs, such as 6 SNPs is used in the method of the present invention.

In such embodiments, the genotype status of a plurality of markers and/or haplotypes is determined in an individual, and the status of the individual compared with the population frequency of the associated variants, or the frequency of the variants in clinically healthy subjects, such as age-matched and sex-matched subjects.

Thus, in one embodiment of the present invention relates the mammal to a human infant selected from the group consisting of a boy and a girl.

In some embodiments of the present invention is said infant less than 2 months old, such as less than 6 weeks old, such as less than 4 weeks old, such as less than 2 weeks old, such as less than 1 week old, such as less than 4 days old, such as less than 2 days old, such as less than one day old.

Methods known in the art, such as multivariate analyses or joint risk analyses, may subsequently be used to determine the overall risk conferred based on the genotype status at the multiple loci. Assessment of risk based on such analysis may subsequently be used in the methods and kits of the invention, as described herein. As described in the above, the haplotype block structure of the human genome has the effect that a large number of variants (markers and/or haplotypes) in linkage disequilibrium with the variant originally associated with a disease or trait may be used as surrogate markers for assessing association to the disease or trait.

The number of such surrogate markers will depend on factors such as the historical recombination rate in the region, the mutational frequency in the region (i.e., the number of polymorphic sites or markers in the region), and the extent of LD (size of the LD block) in the region.

These markers are usually located within the physical boundaries of the LD block or haplotype block in question as defined using the methods described herein, or by other methods known to the person skilled in the art.

However, sometimes marker and haplotype association is found to extend beyond . the physical boundaries of the haplotype block as defined. Such markers and/or haplotypes may in those cases be also used as surrogate markers and/or haplotypes for the markers and/or haplotypes physically residing within the haplotype block as defined. -

As a consequence, markers and haplotypes in LD (typically characterized by r² greater than 0.1, such as r² greater than 0.2, including r² greater than 0.3, also including r² greater than 0.4) with the markers and haplotypes of the present invention are also within the scope of the invention, even if they are physically located beyond the boundaries of the haplotype block as defined.

This includes markers that are described herein but may also include other markers that are in strong LD (characterized by r² greater than 0.1 or 0.2 and/or | D'| > 0.8) with one or more of the markers listed in the present application.

For the SNP markers described herein, the opposite allele to the allele found to be in excess in patients (at-risk allele) is found in decreased frequency in pyloric stenosis. These markers and hapiotypes in LD and/or comprising such markers, are thus protective for pyloric stenosis, i.e. they confer a decreased risk or susceptibility of individuals carrying these markers and/or hapiotypes developing pyloric stenosis.

Certain variants of the present invention, including certain hapiotypes comprise, in some cases, a combination of various genetic markers, e.g., SNPs and

microsatellites. Detecting hapiotypes can be accomplished by methods known in the art and/or described herein for detecting sequences at polymorphic sites.

Furthermore, association between certain hapiotypes or sets of markers and disease phenotype can be verified using standard techniques. A representative example of a simple test for association would be a Fisher-exact test on a two by two table.

Thus in one embodiment of the present invention is the predetermined expression level the expression level from an infant not suffering from pyloric stenosis.

In another embodiment is the comparison of expression levels is done at a predefined statistical significance.

Diagnostic and screening methods

In certain embodiments, the present invention pertains to methods of diagnosing, or aiding in the diagnosis of, pyloric stenosis or a susceptibility to pyloric stenosis, by detecting particular alleles at genetic markers that appear more frequently in pyloric stenosis subjects or subjects who are susceptible to pyloric stenosis. In particular embodiments, the invention is a method of determining a

susceptibility to pyloric stenosis by detecting at least one allele of at least one polymorphic marker (e.g., the markers described herein). In other embodiments, the invention relates to a method of diagnosing a susceptibility to pyloric stenosis by detecting at least one allele of at least one polymorphic marker. The present invention describes methods whereby detection of particular alleles of particular markers or haplotypes is indicative of a susceptibility to pyloric stenosis.

The present invention pertains in some embodiments to methods of clinical applications of diagnosis, e.g., diagnosis performed by a medical professional. -

In other embodiments, the invention pertains to methods of diagnosis or determination of a susceptibility performed by a layman.

The layman can be the customer of a genotyping service. The layman may also be a genotype service provider, who performs genotype analysis on a DNA sample from an individual, in order to provide service related to genetic risk factors for particular traits or diseases, based on the genotype status of the individual (i.e., the. customer). Recent technological advances in genotyping technologies, including high- throughput genotyping of SNP markers, such as Molecular Inversion Probe arrays technology (e.g., Affymetrix GeneChip), and BeadArray Technologies (e.g., Illumina GoldenGate and Infinium assays) have made it possible for individuals to have their own genome assessed for up to one million SNPs simultaneously, at relatively little cost.

The resulting genotype information, which can be made available to the individual, can be compared to information about disease or trait risk associated with various SNPs, including information from public literature and scientific publications.

The diagnostic application of disease-associated alleles as described herein, can thus for example be performed by the individual, through analysis of his/her genotype data, by a health professional based on results of a clinical test, or by a third party, including the genotype service provider. The third party may also be service provider who interprets genotype information from the customer to provide service related to specific genetic risk factors, including the genetic markers described herein. In other words, the diagnosis or determination of a susceptibility of genetic risk can be made by health

5 professionals, genetic counselors, third parties providing genotyping service, third parties providing risk assessment service or by the layman (e.g., the individual), based on information about the genotype status of an individual and knowledge about the risk conferred by particular genetic risk factors (e.g., particular SNPs). In the present context, the term "diagnosing", "diagnose a susceptibility" and 0 "determine a susceptibility" is meant to refer to any available diagnostic method, including those mentioned above.

In certain embodiments, a sample containing genomic DNA from an individual is collected. Such sample can for example be a buccal swab, a saliva sample, a 5 blood sample, or other suitable samples containing genomic DNA, as described further herein.

The genomic DNA is then analyzed using any common technique available to the skilled person, such as high-throughput array technologies. Results from such genotyping are stored in a convenient data storage unit, such as a data carrier, including computer databases, data storage disks, or by other convenient data ^^torage means. database is an object database, a

re ationa ata ase or a post-relational database. The genotype data is subsequently analyzed for the presence of certain variants known to be susceptibility variants for a particular human condition, such as the genetic variants described herein. Genotype data can be retrieved from the data storage unit using any convenient data query method. Calculating risk conferred by a particular genotype for the individual can be based on comparing the genotype of the individual to previously determined risk (expressed as a relative risk (RR) or and odds ratio (OR), for example) for the genotype, for example for an heterozygous carrier of an at-risk variant for a particular disease or trait (such as pyloric stenosis). The calculated risk for the individual can be the relative risk for a person, or for a specific genotype of a person, compared to the average population with matched gender and ethnicity.

The average population risk can be expressed as a weighted average of the risks of different genotypes, using results from a reference population, and the appropriate calculations to calculate the risk of a genotype group relative to the population can then be performed.

Alternatively, the risk for an individual is based on a comparison of particular genotypes, for example heterozygous carriers of an at-risk allele of a marker compared with non-carriers of the at-risk allele. Using the population average may in certain embodiments be more convenient, since it provides a measure which is easy to interpret for the user, i.e. a measure that gives the risk for the individual, based on his/her genotype, compared with the average in the population. The calculated risk estimated can be made available to the customer via a website, preferably a secure website.

In certain embodiments, a service provider will include in the provided service all of the steps of isolating genomic DNA from a sample provided by the customer, performing genotyping of the isolated DNA, calculating genetic risk based on the genotype data, and report the risk to the customer.

In some other embodiments, the service provider will include in the service the interpretation of genotype data for the individual, i.e., risk estimates for particular genetic variants based on the genotype data for the individual. In some other embodiments, the service provider may include service that includes genotyping service and interpretation of the genotype data, starting from a sample of isolated DNA from the individual (the customer).

Overall risk for multiple risk variants can be performed using standard

methodology. For example, assuming a multiplicative model, i.e. assuming that the risk of individual risk variants multiply to establish the overall effect, allows for a straight-forward calculation of the overall risk for multiple markers.

In addition, in certain other embodiments, the present invention pertains to methods of diagnosing, or aiding in the diagnosis of, a decreased susceptibility to pyloric stenosis, by detecting particular genetic marker alleles or haplotypes that appear less frequently in pyloric stenosis patients than in individual not diagnosed with pyloric stenosis or in the general population. As described and exemplified herein are associated with pyloric stenosis. In one embodiment, the marker allele or haplotype is one that confers a significant risk or susceptibility to pyloric stenosis.

In another embodiment, the invention relates to a method of diagnosing a susceptibility to pyloric stenosis in a human individual, the method comprising determining the presence or absence of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual.

In another embodiment, the invention pertains to methods of diagnosing a susceptibility to pyloric stenosis in a human individual, by screening for at least one marker allele or haplotype. ·-

In another embodiment, the marker allele or haplotype is more frequently present in a subject having, or who is susceptible to, pyloric stenosis (affected), as compared to the frequency of its presence in a healthy subject (control, such as population controls).

In certain embodiments, the significance of association of the at least one marker allele or haplotype is characterized by a p value < 0.05. In other embodiments, the significance of association is characterized by smaller p-values, such as < 0.01, <0.001, <0.0001, <0.00001, <0.000001, <0.0000001, <0.00000001 or <0.000000001.

In these embodiments, the presence of the at least one marker allele or haplotype is indicative of a susceptibility to pyloric stenosis. These diagnostic methods involve detecting the presence or absence of at least one marker allele or haplotype that is associated with pyloric stenosis.

The haplotypes described herein include combinations of alleles at various genetic markers (e.g., SNPs or other genetic variants). The detection of the particular genetic marker alleles that make up the particular haplotypes can be performed by a variety of methods described herein and/or known in the art.

For example, genetic markers can be detected at the nucleic acid level (e.g., by direct nucleotide sequencing or by other means known to the skilled in the art) or at the amino acid level if the genetic marker affects the coding sequence of a protein encoded by a pyloric stenosis - associated nucleic acid (e.g., by protein sequencing or by immunoassays using antibodies that recognize such a protein). The marker alleles or haplotypes of the present invention correspond to fragments of a genomic DNA sequence associated with pyloric stenosis.

Such fragments encompass the DNA sequence of the polymorphic marker or haplotype in question, but may also include DNA segments in strong LD (linkage disequilibrium) with the marker or haplotype.

In one embodiment, such segments comprises segments in LD with the marker or haplotype (as determined by a value of r² greater than 0.1 and/or | D'| > 0.8). In one embodiment, diagnosis of a susceptibility to pyloric stenosis can be accomplished using hybridization methods. A biological sample from a test subject or individual (a "test sample") of genomic DNA, RNA, cDNA etc. is obtained from a subject suspected of having, being susceptible to, or predisposed for pyloric stenosis (the "test subject").

Thus, in one embodiment of the present invention is the nucleic acid detected in the method of the present invention a DNA, genomic DNA, RNA, cDNA, hnRNA and/or mRNA. The subject can be an adult, child, infant or fetus. The test sample can be from any source that contains genomic DNA, such as a blood sample, sample of amniotic fluid, sample of cerebrospinal fluid, or tissue sample from skin, muscle, buccal or conjunctival mucosa, placenta, gastrointestinal tract or other organs.

A test sample of DNA from fetal cells or tissue can be obtained by appropriate methods, such as by amniocentesis or chorionic villus sampling. The DNA, RNA, or cDNA sample is then examined. The presence of a specific marker allele can be indicated by sequence-specific hybridization of a nucleic acid probe specific for the particular allele. The presence of more than specific marker allele or a specific haplotype can be indicated by using several sequence-specific nucleic acid probes, each being specific for a particular allele.

In one embodiment, a haplotype can be indicated by a single nucleic acid probe that is specific for the specific haplotype (i.e., hybridizes specifically to a DNA strand comprising the specific marker alleles characteristic of the haplotype). A sequence-specific probe can be directed to hybridize to genomic DNA, RNA, or cDNA.

A "nucleic acid probe", as used herein, can be a DNA probe or an RNA probe that hybridizes to a complementary sequence. One of skill in the art would know how to design such a probe so that sequence specific hybridization will occur only if a particular allele is present in a genomic sequence from a test sample.

The invention can also be reduced to practice using any convenient genotyping method, including commercially available technologies and methods for

genotyping particular polymorphic markers.

To diagnose a susceptibility to pyloric stenosis, a hybridization sample can be formed by contacting the test sample containing a pyloric stenosis-associated nucleic acid, such as a genomic DNA sample, with at least one nucleic acid probe. A non-limiting example of a probe for detecting m NA or genomic DNA is a labeled nucleic acid probe that is capable of hybridizing to mRNA or genomic DNA sequences described herein.

The nucleic acid probe can be, for example, a full- length nucleic acid molecule, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length that is sufficient to specifically hybridize under stringent conditions to appropriate mRNA or genomic DNA.

For example, the nucleic acid probe can comprise all or a portion of a nucleotide sequence comprising the markers listed herein.

Genes or fragments thereof, as described herein, optionally comprising at least one allele of a marker described herein, or at least one haplotype described herein, or the probe can be the complementary sequence of such a sequence.

Hybridization can be performed by methods well known to the person skilled in the art. In one embodiment, hybridization refers to specific hybridization, i.e., hybridization with no mismatches (exact hybridization). In one embodiment, the hybridization conditions for specific hybridization are high stringency.

Specific hybridization, if present, is detected using standard methods. If specific hybridization occurs between the nucleic acid probe and the nucleic acid in the test sample, then the sample contains the allele that is complementary to the nucleotide that is present in the nucleic acid probe.

The process can be repeated for any markers of the present invention, or markers that make up a haplotype of the present invention, or multiple probes can be used concurrently to detect more than one marker alleles at a time.

It is also possible to design a single probe containing more than one marker alleles of a particular haplotype (e.g., a probe containing alleles complementary to 2, 3, 4, 5 or all of the markers that make up a particular haplotype). Detection of the particular markers of the haplotype in the sample is indicative that the source of the sample has the particular haplotype (e.g., a haplotype) and therefore is susceptible to pyloric stenosis. In one preferred embodiment, a method utilizing a detection oligonucleotide probe comprising a fluorescent moiety or group at its 3' terminus and a quencher at its 5' terminus, and an enhancer oligonucleotide, is employed.

The fluorescent moiety can be Gig Harbor Green or Yakima Yellow, or other suitable fluorescent moieties. The detection probe is designed to hybridize to a short nucleotide sequence that includes the SIMP polymorphism to be detected. Preferably, the SNP is anywhere from the terminal residue to -6 residues from the 3' end of the detection probe. The enhancer is a short oligonucleotide probe which hybridizes to the DNA template 3' relative to the detection probe. The probes are designed such that a single nucleotide gap exists between the detection probe and the enhancer nucleotide probe when both are bound to the template. The gap creates a synthetic abasic site that is recognized by an endonuclease, such as Endonuclease IV. The enzyme cleaves the dye off the fully complementary detection probe, but cannot cleave a detection probe containing a mismatch.

Thus, by measuring the fluorescence of the released fluorescent moiety, * assessment of the presence of a particular allele defined by nucleotide sequence of the detection probe can be performed. The detection probe can be of any suitable size, although preferably the probe is relatively short. In one embodiment, the probe is from 5-100 nucleotides in length. In another embodiment, the probe is from 10-50 nucleotides in length, and in another embodiment, the probe is from 12-30 nucleotides in length. Other lengths of the probe are possible and within scope of the skill of the average person skilled in the art.

In a preferred embodiment, the DNA template containing the SNP polymorphism is amplified by Polymerase Chain Reaction (PCR) prior to detection. In such an embodiment, the amplified DNA serves as the template for the detection probe and the enhancer probe.

Certain embodiments of the detection probe, the enhancer probe, and/or the primers used for amplification of the template by PCR include the use of modified bases, including modified A and modified G. The use of modified bases can be useful for adjusting the melting temperature of the nucleotide molecule (probe and/or primer) to the template DNA, for example for increasing the melting temperature in regions containing a low percentage of G or C bases, in which modified A with the capability of forming three hydrogen bonds to its

complementary T can be used, or for decreasing the melting temperature in regions containing a high percentage of G or C bases, for example by using modified G bases that form only- two hydrogen bonds to their complementary C base in a double stranded DNA molecule. In a preferred embodiment, modified bases are used in the design of the detection nucleotide probe. Any modified base known to the skilled person can be selected in these methods, and the selection of suitable bases is well within the scope of the skilled person based on the teachings herein and known bases available from commercial sources as known to the skilled person.

Additionally, or alternatively, a peptide nucleic acid (PNA) probe can be used in addition to, or instead of, a nucleic acid probe in the hybridization methods described herein. A PNA is a DNA mimic having a peptide-like, inorganic backbone, such as N-(2-aminoethyl)glycine units, with an organic base (A, G, C, T or U) attached to the glycine nitrogen via a methylene carbonyl linker. The PNA probe can be designed to specifically hybridize to a molecule in a sample suspected of containing one or more of the marker alleles or haplotypes that are associated with pyloric stenosis. Hybridization of the PNA probe is thus diagnostic for pyloric stenosis or a susceptibility to pyloric stenosis.

Probes can contain one or more of the backbone nucleic acids be substituted by one or more LNAs, TINAs or other nucleic acid analogues to facilitate the optimal assay. In one embodiment of the invention, a test sample containing genomic DNA obtained from the subject is collected and the polymerase chain reaction (PCR) is used to amplify a fragment comprising one or more markers or haplotypes of the present invention.

As described herein, identification of a particular marker allele or haplotype associated with pyloric stenosis, can be accomplished using a variety of methods (e.g., sequence analysis, analysis by restriction digestion, specific hybridization, single stranded conformation polymorphism assays (SSCP), electrophoretic analysis, etc.).

In another embodiment, diagnosis is accomplished by expression analysis, for example by using quantitative PCR (kinetic thermal cycling). This technique can, for example, utilize commercially available technologies, such as TaqMan®.

The technique can assess the presence of an alteration in the expression or composition of a polypeptide or splicing variant(s) that is encoded by a nucleic acid associated with pyloric stenosis. Further, the expression of the variant(s) can be quantified as physically or functionally different.

In another method of the invention, analysis by restriction digestion can be used to detect a particular allele if the allele results in the creation or elimination of a restriction site relative to a reference sequence. Restriction fragment length polymorphism (RFLP) analysis can be conducted, e.g., as described in Current Protocols in Molecular Biology, supra. The digestion pattern of the relevant DNA fragment indicates the presence or absence of the particular allele in the sample. Sequence analysis can also be used to detect specific alleles or haplotypes associated with pyloric stenosis. Therefore, in one embodiment, determination of the presence or absence of a particular marker alleles or haplotypes comprises sequence analysis of a test sample of DNA or RNA obtained from a subject or individual. PCR or other appropriate methods can be used to amplify a portion of a nucleic acid associated with pyloric stenosis, and the presence of a specific allele can then be detected directly by sequencing the polymorphic site (or multiple polymorphic sites in a haplotype) of the genomic DNA in the sample.

Allele-specific oligonucleotides can also be used to detect the presence of a particular allele in a nucleic acid associated with pyloric stenosis, through the use of dot-blot hybridization of amplified oligonucleotides with allele-specific

oligonucleotide (ASO) probes.

An "allele-specific oligonucleotide" (also referred to herein as an "allele-specific oligonucleotide probe") is an oligonucleotide of approximately 10- 50 base pairs or. approximately 15-30 base pairs, that specifically hybridizes to a nucleic acid associated with pyloric stenosis, and which contains a specific allele at a

polymorphic site (e.g., a marker or haplotype as described herein).

An allele-specific oligonucleotide probe that is specific for one or more particular a nucleic acid associated with pyloric stenosis can be prepared using standard methods.

PCR can be used to amplify the desired region. The DNA containing the amplified region can be dot-blotted using standard methods, and the blot can be contacted with the oligonucleotide probe. The presence of specific hybridization of the probe to the amplified region can then be detected. Specific hybridization of an allele- specific oligonucleotide probe to DNA from the subject is indicative of a specific allele at a polymorphic site associated with cancer, including pyloric stenosis.

With the addition of such analogs as locked nucleic acids (LNAs), the size of primers and probes can be reduced to as few as 8 bases. LNAs are a novel class of bicyclic DNA analogs in which the T and 4' positions in the furanose ring are joined via an O-methylene (oxy-LNA), S-methylene (thio-LNA), or amino methylene (amino-LNA) moiety.

Common to all of these LNA variants is an affinity toward complementary nucleic acids, which is by far the highest reported for a DNA analog. For example, particular all oxy-LNA nonamers have been shown to have melting temperatures (Tm) of 64°C and 74°C when in complex with complementary DNA or RNA, respectively, as opposed to 28°C for both DNA and RNA for the corresponding DNA nonamer.

Substantial increases in Tm are also obtained when LNA monomers are used in combination with standard DNA or RNA monomers. For primers and probes, depending on where the LNA monomers are included (e.g., the 3' end, the 5' end, or in the middle), the Tm could be increased considerably.

In another embodiment, arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from a subject, can be used to identify polymorphisms in a nucleic acid associated with pyloric stenosis. For example, an oligonucleotide array can be used. Oligonucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. These oligonucleotide arrays, also described as "Genechips™," have been generally described in the art.

These arrays can generally be produced using mechanical synthesis methods or light directed synthesis methods that incorporate a combination of

photolithographic methods and solid phase oligonucleotide synthesis methods, or by other methods known to the person skilled in the art.

Other methods of nucleic acid analysis that are available to those skilled in the art can be used to detect a particular allele at a polymorphic site associated with pyloric stenosis.

In another embodiment of the invention, diagnosis of pyloric stenosis or a susceptibility to pyloric stenosis can be made by examining expression and/or composition of a polypeptide encoded by a nucleic acid associated with pyloric stenosis in those instances where the genetic marker(s) or haplotype(s) of the present invention result in a change in the composition or expression of the polypeptide. Thus, diagnosis of a susceptibility to pyloric stenosis can be made by examining expression and/or composition of one of these polypeptides, or another polypeptide encoded by a nucleic acid associated with pyloric stenosis, in those instances where the genetic marker or haplotype of the present invention results in a change in the composition or expression of the polypeptide.

The haplotypes and markers of the present invention that show association* to pyloric stenosis may play a role through their effect on one or more of these nearby genes. Possible mechanisms affecting these genes include, e.g., effects on transcription, effects on RNA splicing, alterations in relative amounts of alternative splice forms of mR A, effects on RNA stability, effects on transport from the nucleus to cytoplasm, and effects on the efficiency and accuracy of translation.

Thus, in another embodiment, the variants (markers or haplotypes) of the invention showing association to pyloric stenosis affect the expression of a nearby gene. It is well known that regulatory element affecting gene expression may be located tenths or even hundreds of kilobases away from the promoter region of a gene. By assaying for the presence or absence of at least one allele of at least one polymorphic marker of the present invention, it is thus possible to assess the expression level of such nearby genes.

It is thus contemplated that the detection of the markers or haplotypes of the present invention can be used for assessing expression for one or more of the genes listed herein.

A variety of methods can be used for detecting protein expression levels, including enzyme linked immunosorbent assays (ELISA), Western blots,

immunoprecipitations and immunofluorescence. A test sample from a subject is assessed for the presence of an alteration in the expression and/or an alteration in composition of the polypeptide encoded by a nucleic acid associated with pyloric stenosis. An alteration in expression of a polypeptide encoded by a nucleic acid associated with pyloric stenosis can be, for example, an alteration in the quantitative polypeptide expression (i.e., the amount of polypeptide produced). An alteration in the composition of a polypeptide encoded by a nucleic acid associated with pyloric stenosis is an alteration in the qualitative polypeptide expression (e.g., expression of a mutant polypeptide or of a different splicing variant).

In one embodiment, diagnosis of a susceptibility to pyloric stenosis is made by detecting a particular splicing variant encoded by a nucleic acid associated with pyloric stenosis, or a particular pattern of splicing variants.

Both such alterations (quantitative and qualitative) can also be present. An "alteration" in the polypeptide expression or composition, as used herein, refers to an alteration in expression or composition in a test sample, as compared to the expression or composition of the polypeptide in a control sample.

A control sample is a sample that corresponds to the test sample (e.g., is from the same type of cells), and is from a subject who is not affected by, and/or who does not have a susceptibility to, pyloric stenosis. In one embodiment, the control sample is from a subject that does not possess a marker allele or haplotype as described herein.

Similarly, the presence of one or more different splicing variants in the test sample, or the presence of significantly different amounts of different splicing variants in the test sample, as compared with the control sample, can be indicative of a susceptibility to pyloric stenosis.

An alteration in the expression or composition of the polypeptide in the test sample, as compared with the control sample, can be indicative of a specific allele in the instance where the allele alters a splice site relative to the reference in the control sample. Various means of examining expression or composition of a polypeptide encoded by a nucleic acid are known to the person skilled in the art and can be used, including spectroscopy, colorimetry, electrophoresis, isoelectric focusing, and immunoassays. For example, in one embodiment, an antibody (e.g., an antibody with a detectable label) that is capable of binding to a polypeptide encoded by a nucleic acid associated with pyloric stenosis can be used. Antibodies can be polyclonal or monoclonal. An intact antibody, or a fragment thereof (e.g., Fv, Fab, Fab', F(ab')2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.

Examples of indirect labeling include detection of a primary antibody using a labeled secondary antibody (e.g., a fluorescently-labeled secondary antibody) and end-labeling of a DNA probe with biotin such that it can be detected with

- fluorescently-labeled streptavidin.

In one embodiment of this method, the level or amount of polypeptide encoded by a nucleic acid associated with pyloric stenosis in a test sample is compared with the level or amount of the polypeptide in a control sample. A level or amount of the polypeptide in the test sample that is higher or lower than the level or amount of the polypeptide in the control sample, such that the difference is statistically significant, is indicative of an alteration in the expression of the polypeptide encoded by the nucleic acid, and is diagnostic for a particular allele or haplotype responsible for causing the difference in expression.

Alternatively, the composition of the polypeptide in a test sample is compared with the composition of the polypeptide in a control sample.

In another embodiment, both the level or amount and the composition of the polypeptide can be assessed in the test sample and in the control sample.

In another embodiment, the diagnosis of a susceptibility to pyloric stenosis is made by detecting at least one marker or haplotypes of the present invention in combination with an additional protein-based, NA-based or DNA-based assay. Thus, in one embodiment of the present invention is the detection accomplished by sequencing, mini-sequencing, hybridization, restriction fragment analysis, oligonucleotide ligation assay, or allele specific PCR. Kits

Kits useful in the methods of the invention comprise components useful in any of the methods described herein, including for example, primers for nucleic acid amplification, hybridization probes, restriction enzymes (e.g., for RFLP analysis), allele-specific oligonucleotides, antibodies that bind to an altered polypeptide encoded by a nucleic acid of the invention as described herein (e.g., a genomic segment comprising at least one polymorphic marker and/or haplotype of the present invention) or to a non-altered (native) polypeptide encoded by a nucleic acid of the invention as described herein, means for amplification of a nucleic acid associated with pyloric stenosis, means for analyzing the nucleic acid sequence of a nucleic acid associated with pyloric stenosis, means for analyzing the amino acid sequence of a polypeptide encoded by a nucleic acid associated with pyloric stenosis, etc.

The kits can for example include necessary buffers, nucleic acid primers for amplifying nucleic acids of the invention (e.g., one or more of the polymorphic markers as described herein), and reagents for allele-specific detection of the fragments amplified using such primers and necessary enzymes (e.g., DNA polymerase). Additionally, kits can provide reagents for assays to be used in combination with the methods of the present invention, e.g., reagents for use with pyloric stenosis diagnostic assays.

In one embodiment, the invention is a kit for assaying a sample from a subject to detect the presence of a pyloric stenosis or a susceptibility to pyloric stenosis in a subject, wherein the kit comprises reagents necessary for selectively detecting at least one allele of at least one polymorphism of the present invention in the genome of the individual.

In a particular embodiment, the reagents comprise at least one contiguous oligonucleotide that hybridizes to a fragment of the genome of the individual comprising at least one polymorphism of the present invention. In another embodiment, the reagents comprise at least one pair of

oligonucleotides that hybridize to opposite strands of a genomic segment obtained from a subject, wherein each oligonucleotide primer pair is designed to selectively amplify a fragment of the genome of the individual that includes at least one polymorphism.

In yet another embodiment the fragment is at least 20 base pairs in size. Such oligonucleotides or nucleic acids (e.g., oligonucleotide primers) can be designed using portions of the nucleic acid sequence flanking polymorphisms (e.g., SNPs or microsatellites) that are indicative of pyloric stenosis.

In another embodiment, the kit comprises one or more labeled nucleic acids capable of allele-specific detection of one or more specific polymorphic markers or haplotypes associated with pyloric stenosis, and reagents for detection of the label. Suitable labels include, e.g., a radioisotope, a fluorescent label, an enzyme label, an enzyme co-factor label, a magnetic label, a spin label, an epitope label.

In particular embodiments, the polymorphic marker or haplotype to be detected by the reagents of the kit comprises one or more markers, two or more markers, three or more markers, four or more markers or five or more markers listed herein.

In another embodiment, the marker or haplotype to be detected comprises at least one marker from the group of markers in strong linkage disequilibrium, as defined by values of r² greater than 0.2, to at least one of the group of markers consisting of the markers listed in the tables.

In yet another embodiment, the marker or haplotype to be detected comprises at least one marker selected from the group of markers consisting of markers SNPs rsll712066, rs29784, rs573872, rsll216185, rsl208285, rs2228671,

rsl2721025 and SNPs in linkage disequilibrium therewith.

In one preferred embodiment, the kit for detecting the markers of the invention comprises a detection oligonucleotide probe, that hybridizes to a segment of template DNA containing a SNP polymorphisms to be detected, an enhancer oligonucleotide probe and an endonuclease.

The detection oligonucleotide probe comprises a fluorescent moiety or group at its 3' terminus and a quencher at its 5' terminus, and an enhancer oligonucleotide is employed.

The fluorescent moiety can be Gig Harbor Green or Yakima Yellow, or other suitable fluorescent moieties. The detection probe is designed to hybridize to a short nucleotide sequence that includes the SNP polymorphism to be detected.

Preferably, the SNP is anywhere from the terminal residue to -6 residues from the 3' end of the detection probe. The enhancer is a short oligonucleotide probe which hybridizes to the DNA template 3' relative to the detection probe. The probes are designed such that a single nucleotide gap exists between the detection probe and the enhancer nucleotide probe when both are bound to the template.

The gap creates a synthetic abasic site that is recognized by an endonuclease, such as Endonuclease IV. The enzyme cleaves the dye off the fully complementary detection probe, but cannot cleave a detection probe containing a mismatch.

Thus, by measuring the fluorescence of the released fluorescent moiety, assessment of the presence of a particular allele defined by nucleotide sequence of the detection probe can be performed.

The detection probe can be of any suitable size, although preferably the probe is relatively short. In one embodiment, the probe is from 5-100 nucleotides in length. In another embodiment, the probe is from 10-50 nucleotides in length, and in another embodiment, the probe is from 12-30 nucleotides in length. Other lengths of the probe are possible and within scope of the skill of the average person skilled in the art. In a preferred embodiment, the DNA template containing the SNP polymorphism is amplified by Polymerase Chain Reaction (PCR) prior to detection, and primers for such amplification are included in the reagent kit. In such an embodiment, the amplified DNA serves as the template for the detection probe and the enhancer probe.

Certain embodiments of the detection probe, the enhancer probe, and/or the primers used for amplification of the template by PCR include the use of modified bases, including modified A and modified G.

The use of modified bases can be useful for adjusting the melting -temperature of the nucleotide molecule (probe and/or primer) to the template DNA,-for example for increasing the melting temperature in regions containing a low percentage of G or C bases, in which modified A with the capability of forming three hydrogen bonds to its complementary T can be used, or for decreasing the melting temperature in regions containing a high percentage of G or C bases, for example by using modified G bases that form only two hydrogen bonds to their

complementary C base in a double stranded DNA molecule.

In a preferred embodiment, modified bases are used in the design of the detection nucleotide probe. Any modified base known to the skilled person can be selected in these methods, and the selection of suitable bases is well within the scope of the skilled person based on the teachings herein and known bases available from commercial sources as known to the skilled person.

In one of such embodiments, the presence of the marker or haplotype is indicative of a susceptibility (increased susceptibility or decreased susceptibility) to pyloric stenosis.

In another embodiment, the presence of the marker or haplotype is indicative of response to a pylorics stenosis therapeutic agent. ^ln another embodiment, the presence of the marker or haplotype is indicative of pyloric stenosis prognosis. Thus relates one embodiment of the present invention to a diagnostic kit and/or a research kit, comprising at least one combination of probes for detecting at least one of the haplotypes of described herein and/or probes for determining the expression level the genes listed herein.

5

Nucleic Acids and Polypeptides

The nucleic acids and polypeptides described herein can be used in methods and kits of the present invention, as described in the above. An "isolated" nucleic acid molecule, as used herein, is one that is separated from nucleic acids that normally 10 flank the gene or nucleotide sequence (as in genomic sequences) and/or has been completely or partially purified from other transcribed sequences (e.g., as in an NA library).

For example, an isolated nucleic acid of the invention can be substantially isolated 15 with respect to the complex cellular milieu in which it naturally occurs, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. In some instances, the isolated material will form part of a composition (for example, a crude extract containing other substances), buffer system or reagent mix. In other circumstances, the 20 material can be purified to essential homogeneity, for example as determined by polyacrylamide gel electrophoresis (PAGE) or column chromatography (e.g., HPLC).

An isolated nucleic acid molecule of the invention can comprise at least about 25 50%, at least about 80% or at least about 90% (on a molar basis) of all

macromolecular species present. With regard to genomic DNA, the term "isolated" also can refer to nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated.

30 For example, the isolated nucleic acid molecule can contain less than about 250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb, 25 kb, 10 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of the nucleotides that flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid molecule is derived. The nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated. Thus, recombinant DNA contained in a vector is included in the definition of "isolated" as used herein. Also, isolated nucleic acid molecules include recombinant DNA molecules in heterologous host cells or heterologous organisms, as well as partially or substantially purified DNA molecules in solution. "Isolated" nucleic acid molecules also encompass in vivo and in vitro RNA transcripts of the DNA molecules of the - present invention.

An isolated nucleic acid molecule or nucleotide sequence can include a nucleic acid molecule or nucleotide sequence that is synthesized chemically or by recombinant means. Such isolated nucleotide sequences are useful, for example, in the manufacture of the encoded polypeptide, as probes for isolating homologous sequences (e.g., from other mammalian species), for gene mapping (e.g., by in situ hybridization with chromosomes), or for detecting expression of the gene in tissue (e.g., human tissue), such as by Northern blot analysis or other

hybridization techniques.

The invention also pertains to nucleic acid molecules that hybridize under high stringency hybridization conditions, such as for selective hybridization, to a nucleotide sequence described herein (e.g., nucleic acid molecules that specifically hybridize to a nucleotide sequence containing a polymorphic site associated with a marker or haplotype described herein).

Such nucleic acid molecules can be detected and/or isolated by allele- or sequence-specific hybridization (e.g., under high stringency conditions).

Stringency conditions and methods for nucleic acid hybridizations are well known to the skilled person.

The percent identity of two nucleotide or amino acid sequences can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence). The nucleotides or amino acids at corresponding positions are then compared, and the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions/total # of positions x 100). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, of the length of the reference sequence.

The actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., NBLAST) can be used. See the website on the world wide web at ncbi.nlm.nih.gov. In one embodiment, parameters for sequence comparison can be set at score=100,,wordlength = 12, or can be varied (e.g., W=5 or W=20). The present invention also provides isolated nucleic acid molecules that contain a fragment or portion that hybridizes under highly stringent conditions to a nucleic acid that comprises, or consists of, a nucleotide sequence comprising the polymorphic markers listed herein; or a nucleotide sequence comprising, or consisting of, the complement of the nucleotide sequence of a nucleotide sequence comprising the polymorphic markers listed herein, wherein the nucleotide sequence comprises at least one polymorphic allele contained in the markers and haplotypes described herein.

The nucleic acid fragments of the invention are at least about 15, at least about 18, 20, 23 or 25 nucleotides, and can be 30, 40, 50, 100, 200, 500, 1000, 10,000 or more nucleotides in length.

The nucleic acid fragments of the invention are used as probes or primers in assays such as those described herein. "Probes" or "primers" are oligonucleotides that hybridize in a base- specific manner to a complementary strand of a nucleic acid molecule. In addition to DNA and RNA, such probes and primers include polypeptide nucleic acids (PNA). A probe or primer comprises a region of nucleotide sequence that hybridizes to at least about 15, typically about 20-25, and in certain embodiments about 40, 50 or 75, consecutive nucleotides of a nucleic acid molecule. In one embodiment, the probe or primer comprises at least one allele of at least one polymorphic marker or at least one haplotype described herein, or the complement thereof. In particular embodiments, a probe or primer can comprise 100 or fewer nucleotides; for example, in certain embodiments from 6 to 50 nucleotides, or, for example, from 12 to 30 nucleotides.

In other embodiments, the probe or primer is at least 70% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical, to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence.

In another embodiment, the probe or primer is capable of selectively hybridizing to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence. Often, the probe or primer further comprises a label, e.g., a radioisotope, a fluorescent label, an enzyme label, an enzyme co-factor label, a magnetic label, a spin label, an epitope label.

The nucleic acid molecules of the invention, such as those described above, can be identified and isolated using standard molecular biology techniques well known to the skilled person. The amplified DNA can be labeled (e.g., radiolabeled) and used as a probe for screening a cDNA library derived from human cells. The cDNA can be derived from mRNA and contained in a suitable vector.

Corresponding clones can be isolated, DNA can obtained following in vivo excision, and the cloned insert can be sequenced in either or both orientations by art- recognized methods to identify the correct reading frame encoding a polypeptide of the appropriate molecular weight. Using these or similar methods, the polypeptide and the DNA encoding the polypeptide can be isolated, sequenced and further characterized. In general, the isolated nucleic acid sequences of the invention can be used as molecular weight markers on Southern gels, and as chromosome markers that are labeled to map related gene positions. The nucleic acid sequences can also be used to compare with endogenous DNA sequences in patients to identify pyloric stenosis or a susceptibility to pyloric stenosis, and as probes, such as to hybridize and discover related DNA sequences or to subtract out known sequences from a sample (e.g., subtractive

hybridization).

The nucleic acid sequences can further be used to derive primers for genetic fingerprinting, to raise anti-polypeptide antibodies using immunization-techniques, and/or as an antigen to raise anti-DNA antibodies or elicit immune responses. Treatment of pyloric stenosis

The present invention also relates to the treatment of pyloric stenosis in infants.

The inventors have surprisingly found that pyloric stenosis has a link to the cholesterol metabolism and that supplementary cholesterol or any other derivative hereof can be used in the treatment of pyloric stenosis in infants.

Thus relates one aspect of the present invention to the use of cholesterol in the treatment of pyloric stenosis in infants and pharmaceutical compositions comprising cholesterol for treatment of IHPS.

Other means of treatment include any type of medication or supplementary that has direct or indirect influence on the cholesterol metabolism including High- density lipoprotein (HDL), Very High-density lipoprotein (VHDL), Low-density lipopeotein (LDL).

Such medicaments include various hypolipidemic agents such as statins, fibrates, cholesterol absorption inhibitors, nicotinic acid derivatives or bile acid

sequestrants. Example 1 - Common Genetic Variants near MBNL1 and NKX2-5 are Associated with Infantile Hypertrophic Pyloric Stenosis

INTRODUCTION

Infantile Hypertrophic Pyloric Stenosis (IHPS) is a severe condition of infancy in which hypertrophy of the pyloric sphincter smooth muscle leads to obstruction of the gastric-outlet. Symptoms typically appear between 2 and 8 weeks after birth and include non-bilious projectile vomiting that progresses to dehydration, weight loss and hypochloremic, hypokalemic metabolic alkalosis.

Treatment may require correction of electrolyte disturbances and curative surgical incision of the pyloric sphincter muscle. Because vomiting is common in young infants the diagnosis may be delayed and can result in significant morbidity and mortality when access to appropriate care is limited. The incidence of IHPS among whites is 1.5 to 3 per 1000 live births, and it is the most common condition requiring surgery in the first months of life. There is a pronounced male excess in the incidence of IHPS, with affected boys outnumbering girls in a 4 to 1 ratio.

While environmental risk factors such as exposure to erythromycin and bottle feeding have been reported, IHPS also aggregates strongly in families. In a population-based cohort study of almost 2 million children, there was a nearly 200-fold increased risk in monozygotic twins, a 20-fold increased risk among siblings and with heritability estimated to be 87%. IHPS is generally regarded as a complex disease with multiple genetic and environmental factors contributing to disease pathogenesis. Several linkage and candidate gene studies have been conducted, but there are no genetic variants with replicated association findings.

To identify common sequence variants associated with IHPS as a prelude to a better understanding of its biology and as a tool to identify high-risk individuals, we used a genome-wide association study (GWAS) approach. We studied an ethnically homogeneous population from Denmark with linkage of Electronic Medical Records for diagnosis to a biorepository of blood spots from population based Neonatal Screening in Denmark to provide samples for DNA extraction. Controls were drawn from the same population and a large independent sample set of cases and controls was available to replicate positive signals.

METHODS SUBJECTS

Eligible IHPS cases were defined as children who 1) in their first year of life had a pyloromyotomy 2) were singletons 3) did not have any major malformations, and 4) were of Danish ancestry. In addition, we excluded severe pregnancy complications (see supplementary note for details about selection criteria). In the discovery stage, samples from 1,001 cases were selected and successfully genotyped.

The control group consisted of 2,401 non-affected Danish children. Apart from IHPS affection status, the selection criteria were the same as for the cases. For the replication stage, we used 796 cases and 876 controls drawn from the same population using the same case and control definitions as in the discovery stage. The sex distribution was similar in the discovery and replication samples, but replication cases were born an average of 6 years earlier than discovery cases (Figure 3). The study was approved by the Scientific Ethics Committee for the Capital City Region (Copenhagen) and the Danish Data Protection Agency.

GENOTYPING AND QUALITY CONTROL

All samples were drawn from the Danish Newborn Screening Biobank and the Danish National Birth Cohort biobank, both of which are part of the Danish National Biobank. Sampling and genotyping (using the Illumina Human 660W- Quadvl_A chip) was undertaken in two rounds (see supplementary note for details).

In total, genotypes for 559,390 SNPs were released in both genotyping rounds. For the association analysis, we used 523,420 SNPs; the other SNPs were excluded based on a missing rate >5%, deviation from Hardy-Weinberg equilibrium in controls (P< 10-3), minor allele frequency < 1%, or discrepancies (P< 10-7) in allele frequencies between the two genotyping rounds. Genotyping of replication samples was performed on two correlated SNPs at each of the 6 most significantly associated loci from the discovery stage at deCODE Genetics using the Centaurus platform (Nanogen). We regenotyped 147 discovery stage samples and observed 100% concordance in a total of 1604 genotypes.

STATISTICAL ANALYSIS

For the SNPs passing quality control, we used PLINK to test for differences in allele frequencies between cases and controls for the discovery samples overall and stratifying by sex.

P values were corrected by genomic control using estimated genomic inflation factors of 1.06 in the combined discovery data, 1.04 in the analysis restricted to boys and 1.02 in the analysis for girls. We carried out combined analysis of the discovery and replication data using the inverse variance method as implemented in METAL and tested for heterogeneity of the discovery and replication results using the 12 statistic. Using the combined discovery and replication data, we tested for interaction effects between the top six loci by including risk allele count at each locus in a logistic regression model together with pair-wise interaction terms.

RESULTS

To identify IHPS susceptibility loci, we analyzed the association between the disease and 523,420 SNPs in 1,001 cases and 2,401 controls of Danish descent. We identified six loci associated at P< = lxl0^"6 (see Figure 7 and 8 for quantile- quantile and Manhattan plots). To confirm the observed associations we genotyped and tested the most significant SNP at each of the six loci in a replication sample of 796 cases and 876 controls, all of Danish descent. Three loci replicated very clearly with combined p-values < lxlO^"11, two loci showed less pronounced replication but remained suggestive, and one locus failed to replicate (Figure 1). Genotyping of one additional SNP at each locus produced essentially the same results (Figure 4).

On chromosome 3q25.1, the variant rsl 1712066 showed strong association with IHPS (odds ratio, 1.61; P=1.5xl0^"17). Rsll712066 is located 150 kb upstream of the MBNL1 (muscleblind-like 1) gene with a recombination hotspot between the SNP and the gene (Figure 2a).

The closest genes on the other (centromeric) side are AADAC (arylacetamide deacetylase) and SUCNR1 (succinate receptor 1); rsll712066 is located about 250Kb downstream of these genes. The second genome-wide significant variant, rs573872 (odds ratio, 1.41; P=4.3xl0^"12), is located at chromosome 3q25.2 in a gene desert some 1.3Mb downstream of MBNL1. The closest genes to the SNP are C3orf79 (Chromosome 3 open reading frame 79) about 250kb centromeric and SGEF (SH3-containing Guanine Nucleotide Exchange Factor) about 370kb telomeric (Figure 2b). Further centromeric between rs573872 and MBNL1 there are two small genes P2RY1 (purinergic receptor P2Y, G-protein coupled, 1) and RAP2B (RAP2B, member of RAS oncogene family).

The third genome-wide significant variant, rs29784 (odds ratio, 1.42; P=1.5xl0- 15), is located on chromosome 5q35.2 between the BNIP1 (BCL2/adenovirus E1B 19kDa interacting protein 1) gene and the NKX2-5 (NK2 transcription factor related, locus 5) gene in a linkage disequilibrium (LD) block that contains both genes (Figure 2c).

Considering the combined number of risk alleles across the three SNPs, the 8% of children carrying five or six risk alleles were at almost five times higher risk of IHPS compared to the 8% with zero or one alleles (odds ratio, 4.91; 95% confidence interval, 3.59 to 6.71).

Two variants almost achieved nominal significance in the replication stage, suggestive of an association. The first of those, rsl208285 on chromosome 6q23.2, is in the MGC34034 gene encoding a hypothetical protein in an LD block that also contains the TCF21 (transcription factor 21) gene (Figure 8a). Within TCF21, the variant rsl2190287 was recently found to be associated with coronary artery disease (CAD) and TCF21 expression levels (15). Based on data from 60 HapMap Europeans, the IHPS risk (C) allele of rsl208285 is positively correlated with the CAD risk (C) allele of rsl2190287 (r=0.55; r2=0.31). The other potentially associated SNP, rsll216185 on chromosome llq23.3, is an intronic variant in the salt-inducible kinase 3 (SIK3) gene. The LD block containing rsll216185 extends several hundred kb; immediately centromeric of this block is a region with the apolipoprotein genes APOA1, APOC3, APOA4, and APOA5 (Figure 8b).

Finally, while rs2228671 on chromosome 19pl3.2 failed to replicate, there was significant heterogeneity between effect estimates in boys and girls (Figure 5). In the discovery data, rs2228671 was strongly associated in boys, but showed no effect in girls. In the replication study, the effect estimate for boys was in the same direction as in the discovery data, but the estimate for girls was in the opposite direction. R.S2228671 is a synonymous SNP located in the first exon of the low density lipoprotein receptor precursor (LDLR) gene (Figure 8c); the minor allele T (which confers risk of IHPS in our discovery data) has been shown to lower circulating levels of LDL cholesterol and total cholesterol.

For the five other SNPs, there was no indication of heterogeneity of effects between sexes (Figure 5). Also, we found no evidence of interaction between the top three loci (data not shown).

DISCUSSION

In this first GWAS of IHPS, we identified three previously unknown, robust associations between common sequence variants and disease status. Two of these associations represent independent signals 1.6Mb apart on chromosome 3 on either side of MBNL1.

Other genes in the region have roles in modulation of intracellular biochemistry and regulation of platelet aggregation and are not obvious candidates for IHPS. By contrast, MBNL1 is a strong candidate for IHPS.

Members of the muscleblind protein family are important regulators of alternative splicing. It has recently become clear that almost all human multiexon genes undergo alternative splicing. In the early post-natal period, splicing transitions from fetal to adult protein isoforms are essential for the extensive remodeling of muscle tissue that occurs as a part of normal development.

In studies of mouse heart and skeletal muscle development, Mbnll has been shown to control a set of temporally correlated splicing transitions that occur within the first 3 weeks of post-natal life. This coincides with a change in the subcellular distribution of Mbnll from a predominantly cytoplasmic location at post-natal day 2 to a predominantly nuclear location at post-natal day 20.

Additionally, expression levels of Mbnll show distinct temporal changes in the early post-natal period correlated with the splicing transitions.

Disruption of normal MBNL1 gene function is an important mechanism in several RIMA-mediated diseases, most notably myotonic dystrophy (DM). The common form, DM1, is caused by expansion of CTG repeats in the 3' untranslated region of the DMPK (dystrophia myotonica protein kinase) gene. The resulting mRNA contains abnormally expanded CUG repeats (CUGexp) and is retained in nuclear foci. MBNL1 protein binds to CUGexp RNA with high affinity thereby becoming sequestered in nuclear foci and depleted in the nucleoplasm. Loss of MBNL1 has a pivotal role in the pathogenesis of DM1.

Mice deficient in Mbnll show characteristics of myotonic dystrophy. Moreover, the loss of Mbnll leads to misregulation of alternative splicing characteristic of DM1. It has been estimated that loss of Mbnll explains more than 80% of the splicing pathology due to CUGexp.

Our results suggest that MBNL1 may be involved in additional muscle-related disease through mechanisms other than sequestration by toxic RNA. The intriguing observations that 1) IHPS occurs almost exclusively between 2 and 8 weeks after birth, and 2) MBNL1 controls many temporally correlated splicing transitions within the first 3 weeks after birth point to the possible role of misregulation of MBNLl-controlled splicing transitions in the etiology of IHPS.

At chromosome 5q35.2, close to NKX2-5, we found rs29784 to be strongly associated with IHPS. NKX2-5 encodes the homeobox transcription factor NKX2-5, which is essential for normal heart formation and development. In humans, a range of NKX2-5 mutations have been identified that cause congenital heart defects (CHDs). There is substantial heterogeneity in the clinical presentation of persons with NKX2-5 mutations; commonly associated malformations include atrial septal defects with or without atrioventricular block, isolated ventricular septal defects, and tetralogy of Fallot.

Furthermore, in an independent study we have found epidemiological evidence suggesting commonalities in the etiology of IHPS and congenital heart defects (CHD). Using a cohort of all persons born in Denmark in 1977-2008, the incidence rate ratio of IHPS given a CHD diagnosis was 2.61 (95% confidence interval, 2.05 to 3.27) (unpublished results).

These findings warrant further research into the mechanisms whereby NKX2-5 may cause cardiac and smooth muscle overgrowth. In addition to CHD, NKX2-5 is also implicated in myotonic dystrophy; it was recently demonstrated that DM1 is associated with induced NKX2-5 expression in cardiac and skeletal muscle.

Although NKX2-5 is not expressed in adult extracardiac tissues studies of embryonic gut development have shown that NKX2-5 is crucial for the formation of pyloric sphincter muscle tissue. In both chicken and mouse, Nkx2-5 expression occurs in a sharply defined ring of mesenchyme at the junction between the foregut and midgut on specific days of embryonic development.

Furthermore, repression of Nkx2-5 activity in the pyloric sphincter region results in loss of the pyloric sphincter endodermal phenotype; conversely, formation of pyloric sphincter-like epithelium in other parts of the gizzard (the equivalent of the stomach in the chicken) can be induced by ectopic expression of Nkx2-5 via a retroviral vector. Further studies are needed to address the mechanisms whereby NKX2-5 may contribute to the risk of IHPS. Although biopsies of pyloric sphincter muscle tissue would be difficult to obtain, comparison of NKX2-5 expression in such tissue from IHPS patients with that from age-matched controls would be interesting, as might be analysis of NKX2-5 alternative splicing in pyloric sphincter muscle tissue using m NA sequencing. Two variants, rsl208285 on chromosome 6q23.2 and rsll216185 on

chromosome llq23.3, remained suggestive after replication. The variant on 6q is correlated with a risk variant for coronary artery disease providing additional suggestive evidence for a connection between biological mechanisms involved in heart disease and IHPS. The variant on llq lies adjacent to locations where linkage was previously reported for IHPS.

One variant, rs2228671 on 19pl3.2, failed to replicate, but showed heterogeneity in effect between boys and girls. Further study is needed to establish whether these variants contribute to the risk of IHPS. It should be noted that the mean birth year for the cases in the replication study was 1990 compared to 1996 in the discovery case group. The substantial decline in IHPS incidence that occurred in Denmark in the early 1990s is likely to be due to reduced influence of

environmental factors.

We therefore speculate that the influence of genetic factors could have been less pronounced in the replication cases compared to the discovery cases thereby slightly lowering statistical power for replication.

Although the chromosome 11 and 19 loci were not confirmed, the fact that they are in or close to genes affecting serum lipid levels deserves discussion.

Interestingly, two reported risk factors for IHPS, male sex and bottle-feeding, both influence infant serum lipid levels. It has long been known that levels of LDL cholesterol and high density lipoprotein (HDL) cholesterol are on average higher in newborn girls compared to boys.

Also, mean total cholesterol and LDL cholesterol levels are higher in breastfed babies than in bottlefed infants. Furthermore, IHPS is a prominent clinical feature in many reports of the Smith-Lemli-Opitz syndrome, an inborn defect of cholesterol biosynthesis in the gene DHCR7. In one large case series, six of 49 cases with proven DHCR7 mutations had IHPS. Together, these observations suggest that levels of circulating cholesterol in newborns could play a role in the etiology of IHPS and that the protective effects of female sex and of breastfeeding may in part be due to higher cholesterol levels. In conclusion, we conducted a data-driven genome-wide investigation of genetic risk factors for IHPS and identified three independent, strongly associated loci near strong candidate genes involved in regulation of alternative splicing, cardiac muscle development, and embryonic gut development. We also found suggestive evidence for additional loci with roles in coronary artery disease risk and regulation of cholesterol levels. Further investigation of the associations identified here will illuminate the biological mechanisms behind this enigmatic condition and may lead to new approaches for prevention or treatment. Supplementary note

Subjects

IHPS cases were identified based on the Danish National Patient Register, which covers all hospital discharge diagnoses and operations performed since 1977. Eligible cases were defined as children who, in their first year of life, had a pyloromyotomy according to the Danish Classification of Surgical Procedures codes up to December 1995 (International Statistical Classification of Diseases, Eighth Revision (ICD-8) codes 41840, 41841, 44100) and the Nordic Classification of Surgical Procedures codes after January 1996 (ICD-10 codes KJDH60,

KJDH61).

The total number of cases born between 1977 and 2008 and operated within the first year was 3,366 (more than 90% of these were operated within the first two months). We excluded multiple births and major malformations at birth according to the EUROCAT classification (http://www.eurocat-network.eu/). We also excluded the following pregnancy complications: placenta praevia, placental abruption, placental insufficiency, hydramnios, isoimmunization, and

preeclampsia. To ensure a high degree of genetic homogeneity in the genotyped sample, we obtained birthplace information from the Danish Civil Registry, and only included cases who themselves as well as their parents were born in Scandinavia and whose grandparents were not born outside of Northwestern Europe. 1,048 case samples were submitted for genome-wide SNP genotyping, prioritizing most recent birth years. Of these, 1,001 were successfully genotyped. The control group used in this study consisted of 2,401 non-affected children from our ongoing GWAS of preterm delivery, with the same exclusion criteria applied. For replication, we had 796 cases and 876 controls using the same case and control definitions as in the discovery stage.

Sampling, amplification and genotyping

All samples were retrieved from the Danish Newborn Screening Biobank or from the biobank of the Danish National Birth Cohort, both of which are part of the Danish National Biobank. Sampling and genotyping of the discovery stage subjects was undertaken in two rounds. In the first round, 1,901 children were sampled from buffy coat (1,440) or dried blood spot samples (461) as a part of our preterm delivery GWAS. DNA extraction and genotyping was performed at Johns Hopkins University Center for Inherited Disease Research (CIDR).

For 55 samples whole-genome amplification was performed due to low yields of genomic DNA. GWAS genotyping was done with Illumina (Illumina, San Diego, CA, USA) Human 660W-Quadvl_A chip.

In the second round, all IHPS cases as well as 500 extra samples from the preterm delivery GWAS were sampled using two 3mm punches from dried blood spot samples. For these samples, DNA was extracted and whole-genome amplified at Statens Serum Institut using a protocol optimized for dried blood spot samples as previously described. GWAS genotyping was again performed at CIDR using the Illumina Human 660W-Quadvl_A chip.

For replication, we sampled 796 cases and 876 controls using punches of dried blood spot samples. DNA was extracted and whole-genome amplified at Statens Serum Institut. Genotyping of two correlated SNPs at each of the 6 most significantly associated loci was performed at deCODE Genetics using the

Centaurus platform (Nanogen, Bothell, WA, USA).

Example 2 - Plasma lipids and the risk of Infantile Hypertrophic Pyloric Stenosis METHODS SUBJECTS

Samples from Danish subjects correspond to samples in example 1. Additionally we included 129 Swedish cases and 742 controls with DNAs available at

Karolinska Institutet and 738 cases from the United States and 697 controls with DIMA available at the University of Iowa. The plasma measurement study was based on subjects from the Danish National Birth Cohort with umbilical cord blood samples available. There were 46 cases with adequate amounts of plasma, 34 of which also had genotype data available. Controls were sampled among the controls already included in the discovery stage of the genetic study. For each case subject four control subjects were sampled matched on gender and gestational age at birth, making the total number of controls 189. The study was approved by the Scientific Ethics Committee for the Capital City Region

(Copenhagen) and the Danish Data Protection Agency. GENOTYPING AND QUALITY CONTROL

Sampling and genotyping (using the Illumina Human 660W-Quadvl_A chip) was undertaken in two rounds. In total, genotypes for 559,390 SNPs were released in both genotyping rounds. Prior to imputation, we required subjects to have a genotype call rate >97%, and we excluded SNPs based on a missing rate >2%, deviation from Hardy-Weinberg equilibrium in controls (P< 10^~3), minor allele frequency <0.5%, or discrepancies (P< 10 ⁷) in allele frequencies between the two genotyping rounds. We also lifted the genotype data from NCBI build 36 to NCBI build 37 and aligned all genotypes to the forward strand. Finally, we excluded SNPs that did not match known variant positions in the 1,000 Genomes project reference data. The remaining 529,128 SNPs were used for imputation. We re- genotyped 144 discovery stage samples and observed 99.7% concordance between 1002 observed or best-guess imputed discovery stage genotypes and the corresponding replication Imputation

We used a two-step procedure to impute unobserved genotypes using phased haplotypes from the integrated Phase I release of the 1,000 Genomes Project. In a first pre-phasing step, we used SHAPEIT to estimate haplotypes for the IHPS study samples. In a second step, we imputed missing alleles for additional SNPs directly onto these phased haplotypes using IMPUTE2. After a first round of association testing, we re-imputed the most significantly associated regions using the original IMPUTE2 algorithm (i.e. without pre-phasing) for increased accuracy. We chose imputed SNPs or insertion/deletions (indels) with minor allele frequencies of > 1% and SNPTEST proper info value of >0.5 for further analyses.

PLASMA MEASUREMENTS

The umbilical cord plasma samples were drawn from the Danish National Birth Cohort biobank. Aliquots of 40 ul were prepared and spectrophotometric measurements were done using the Roche Cobas c 111 Analyzer yielding measurements of circulating LDL, HDL, and Total Cholesterol, as well as

Triglycerides.

STATISTICAL ANALYSIS

We used SNPTEST to conduct tests for differences in allele dosages between cases and controls under an additive genetic model. These analyses were done for the discovery samples overall and stratified by sex. P values were corrected by genomic control using estimated genomic inflation factors of 1.05 in the combined discovery data, 1.02 in the analysis restricted to boys and 1.03 in the analysis for girls. We carried out combined analysis of the discovery and replication data using the inverse variance method as implemented in METAL and tested for

heterogeneity of the discovery and replication results using the I² statistic. Using the combined discovery and replication data, we tested for interaction effects between the top loci by including risk allele dosage at each locus in a logistic regression model together with pair-wise interaction terms.

We evaluated the association between lipid levels in umbilical cord blood and the risk of IHPS by relative risks (RRs) approximated by odds ratios estimated in a conditional logistic regression using the R software package. The analysis took into account matching by sex and gestational age at birth. P values were obtained using likelihood ratio tests, and all tests were two-sided. We also divided measurements into quartiles to allow for nonlinear association. To avoid ties in this analysis, we added a random number from a Gaussian distribution with mean 0 and variance 10^"20 to each measurement.

RESULTS

To identify IHPS susceptibility loci,* we analyzed the association between the disease and 9,494,370 imputed genetic variants in 1,001 cases and 2,371 controls of Danish descent. Four loci showed after-imputation P values of < lxl0^"7 and were selected for further study. These included two novel loci on chromosomes llq23.3 and 19pl3.2, as well as two already confirmed loci on chromosomes 3q25.1 and 5q35.2. The third known locus on chromosome 3q25.2 was also selected for further study to allow. analyses including all known IHPS loci. The chromosome regions harboring the five selected loci were re-imputed with the original IMPUTE2 algorithm for increased accuracy, and association tests were repeated for these regions. To confirm the associations at these loci, we genotyped a total of seven SNPs in replication samples from Denmark, Sweden, and the United States with a total of 1,663 cases and 2,315 controls. One novel locus (llq23.3) replicated with genomewide significance, the three known loci were confirmed, and one locus failed to replicate (Figure 9).

The variant rsl2721025 yielded the strongest association result (odds ratio, 1.59; P=1.8xl0^"10) at the llq23.3 locus. This SNP is located 301 bases downstream of the Apolipoprotein A-I (APOA1) gene with additional apolipoprotein genes APOC3, APOA4, and APOA5 within 50 kb centromeric (Figure 10). A region of strong linkage disequilibrium extends several hundred kb to the telomeric side; here the variant rs77349713, which is intronic in the salt-inducible kinase 3 (SIK3) gene, also associated with genomewide significance (odds ratio, 1.53; P=1.2xl0^"9). We explored the possible functional impact of the llq23.3 associations by considering all 222 genotyped or imputed variants (SNPs and indels) at the locus with P < 1 x 10^"6. These were all highly correlated with rsl2721025 (r2 between 0.68 and 0.96) and most were intronic in the genes APOA1, SIK3, PAFAH1B2, SIDT2, TAGLN, or PCSK7. Two SNPs were in exons, but both were synonymous. A search of the GWAS catalog did not reveal any associations to other phenotypes. However, 50 SNPs were previously found to associate with levels of total and/or HDL cholesterol with P values down to 4.9 x 10^"7 (results retrieved from

wikigwa.org). For all of these SNPs, the cholesterol lowering allele consistently conferred increased risk of IHPS (Figure 11). To further investigate the role of lipids for disease risk, we measured plasma levels of total, LDL, and HDL cholesterol as well as triglycerides in umbilical cord blood from a set of 46 IHPS cases and 189 controls of Danish ancestry. Table 8 shows RRs of IHPS according to total cholesterol levels in umbilical cord blood plasma. The mean total cholesterol levels for 46 case subjects and 189 matching control subjects were 1.69 mmol/L and 1.95 mmol/L, respectively. The rate of IHPS was inversely and significantly associated with total cholesterol level with a 64% (95% confidence interval -83% to -26%; P=0.005) lower rate per mmol/L. Total cholesterol levels divided into quartiles to allow for a nonlinear association showed a trend of lower RRs from quartile 1 through 4- with a five-fold lower rate in the highest quartile compared to the lowest. Results for LDL and HDL cholesterol and triglycerides were similar, although quartile 3 had the lowest RR for HDL cholesterol and triglycerides (Table 9).

DISCUSSION

In the largest genetic study of IHPS to date, we included more than 2,600 cases and 4,600 controls from three countries and analyzed more than 10 million genetic variants through imputation. We confirmed three known loci and identified a novel genomewide significant locus on chromosome llq23.3 in a region harboring the apolipoprotein (APOA1/C3/A4/A5) gene cluster. The most significant SNP, rsl2721025, is located immediately downstream of APOAl and is directly covered by several different histone modification regions.

APOAl encodes apolipoprotein A-I which is the major protein component of high density lipoprotein (HDL) in plasma, pointing to the importance of plasma lipids for IHPS. More specifically, our results suggest that low levels of plasma cholesterol increase the risk of IHPS in newborns. In support of this hypothesis, rsl2721025 is strongly correlated with SNPs previously found to be associated with levels of circulating cholesterol. For these SNPs, the cholesterol lowering allele consistently conferred increased risk of IHPS. Also, in plasma lipid

measurements from umbilical cord blood, we found that having a low level of total cholesterol at birth was significantly associated with increased risk of developing IHPS; Interestingly, IHPS is a prominent clinical feature in many reports of the Smith-Lemli-Opitz syndrome (SLOS), an inborn defect of cholesterol biosynthesis in the gene DHCR7. In one large case series, six of 49 cases with proven DHCR7 mutations had IHPS. A study of 10 IHPS patients and 8 controls sampled at ages 4 to 6 weeks found no cholesterol metabolism anomalies in isolated IHPS cases, but did find that plasma cholesterol levels were lower in cases compared to controls. The study was however too small for firm statistical conclusions and appears not to have been followed up.

A number of observations that have long puzzled epidemiologists are also consistent with low lipid levels being an important risk factor for IHPS. First, the protective effect of female sex could at least partly be due to higher cholesterol levels, since it is well-known that levels of LDL cholesterol and HDL cholesterol are on average higher in newborn girls compared to boys. Second, the IHPS risk associated with bottle-feeding could in part be caused by insufficient lipid levels since bottle-feeding is known to be associated with lower total and LDL cholesterol levels in infancy. Finally, the recent drop in IHPS incidence observed in many countries coincided temporally with improved nutritional composition of milk formula and also with increasing percentages of baby mothers breastfeeding their infants, suggesting that better nutritional status in infants may have prevented IHPS from developing in a fraction of potential cases.

Functional studies of the pathogenic mechanisms relating low levels of cholesterol to IHPS are clearly needed. Cholesterol has a multitude of biological functions. However, given the deficiencies in enteric innervation seen in pyloric sphincter muscle tissue from IHPS patients, the essential role of cholesterol in nervous system development warrants attention in particular. There is compelling evidence that the cellular membrane contains microdomains (known as lipid rafts) that are enriched in cholesterol and sphingolipids. A number of studies have shown that receptors for different growth factors are localized in lipid rafts and that lipid rafts are important mediators of neurite outgrowth. Thus the availability of cholesterol limits normal nervous system development. In relation to IHPS, a possible pathogenic mechanism could entail low levels of cholesterol resulting in

insufficient lipid raft formation, and thereby delayed or deficient innervation of the pyloric circular muscle layer. The consequent failure of pyloric sphincter relaxation could in turn induce subsequent hypertrophy and gastric outlet obstruction. It is important to note that patients with isolated IHPS generally do not show

manifestations of severe cholesterol deficiency, as seen in e.g. SLOS. It rather appears that the normal post-natal development of pyloric sphincter innervation is particularly sensitive to having cholesterol levels in the lower end of the normal range.

Although the three known genetic loci on chromosomes 3p25.1, 3p25.2, and 5q35.2 were confirmed in combined analysis of the replication data from Sweden and the U.S, there was some heterogeneity in effect estimates (Figure 9). One reason could be that differences between the cohorts in local LD structure could result in weaker correlation between the genotyped SNP and the underlying causal variant at the locus. In the associated regions, the strongest candidate genes for IHPS are MBNLl (3p25.1 and 3p25.2), NKX2-5 (5q35.2) and APOAl (llq23.3).

Functionally these genes have roles in alternative splicing regulation, cardiac muscle development and embryonic gut development, and cholesterol

metabolism, re-emphasizing the complexity of IHPS etiology.

In conclusion, we identified a novel genetic locus besides the ones described in example 1, that robustly associates with IHPS. The findings suggest that the low levels of cholesterol increase the risk of IHPS. We found further support for this hypothesis through plasma lipid measurements in umbilical cord blood. The 25% of newborns with the lowest levels of total cholesterol were at almost five times higher risk compared with the 25% with the highest levels (Table 8). In terms of clinical relevance, our findings suggest that dietary supplementation to ensure normal lipid levels in at risk infants warrants investigation as an intervention possibility aimed at reducing the number of pyloromyotomies in IHPS cases. Tables

Examples of Linkage disequilibrium (top 20) are listed in tables 1-7.

In the first column is the chromosome of the first SNP listed. In the second column is the basepair position of the first SNP listed. In the third column is the name of the first SNP listed.

Columns four to six lists the chromosome position, basepair position and SNP name of the second SNP, respectively.

The last row (row seven) lists the R² value of the two SNPs based on 60 unrelated individuals from the HapMap phase II CEU data set (Tables 1-6) or based on 1000 Genomes imputed allele dosages in 2371 Danish control individuals (Tabled).

Table 1 - LD for rs29784

5 172527914 rs29784 5 172528467 rs39796 0.964588

5 172527914 rs29784 5 172501621 rs2544580 0.893474

5 172527914 rs29784 5 172425198 rs435216 0.888735

5 172527914 rs29784 5 172541833 rs255308 0.837107

5 172527914 rs29784 5 172533355 rsl819985 0.829746

5 172527914 rs29784 5 172510375 rs29794 0.802115

5 172527914 rs29784 5 172538555 rs255309 0.796252

5 172527914 rs29784 5 172540810 rs29782 0.796252

5 172527914 rs29784 5 172537147 rs255312 0.76195

5 172527914 rs29784 5 172503011 rs255307 0.756414

5 172527914 rs29784 5 172459176 rs251230 0.747329

5 172527914 rs29784 5 172545634 rs255315 0.708438

5 172527914 rs29784 5 172551669 rs255322 0.708438

5 172527914 rs29784 5 172551951 rs255324 0.708438

5 172527914 rs29784 5 172539424 rs29783 0.692157

5 172527914 rs29784 5 172546391 rsl94255 0.635806

5 172527914 rs29784 5 172557088 rs7701353 0.56571

5 172527914 rs29784 5 172510009 rs29795 0.5499

5 172527914 rs29784 5 172513472 rs255292 0.5499

5 172527914 rs29784 5 172531924 rs2250766 0.537778 Table 2 - LD for rs573872

3 154954853 rs573872 3 154945730 rs519073 1

3 154954853 rs573872 3 154946071 rs543037 1

3 154954853 rs573872 3 154954853 rs573872 1

3 154954853 rs573872 3 154955513 rs565329 1

3 154954853 rs573872 3 154955703 rs9851769 1

3 154954853 TS573872 3 154955882 rs493799 1

3 154954853 rs573872 - ,3 154956565 rs581425 1

3 154954853 rs573872 3 , 154956910 rs510173 1

3 154954853 rs573872 3 154957104 rs547578 1

3 154954853 rs573872 3 154957148 rs512603 1

3 154954853 rs573872 3 154957334 rs659379 1

3 154954853 rs573872 3 154957682 rs657652 1

3 154954853 rs573872 3 154957803 rs539376 1

3 154954853 rs573872 3 154956230 rs518339 0.925502

3 154954853 rs573872 3 154956486 rsl699525 0.916032

3 154954853 rs573872 3 154958223 rs578788 0.776224

3 154954853 rs573872 3 154954727 rs558023 0.54

3 154954853 rs573872 3 154942090 rs532478 0.34953

3 154954853 rs573872 3 154942032 rs532315 0.339706

3 154954853 rs573872 3 154942530 rs565129 0.339706

3 154954853 rs573872 3 154943589 rsl532795 0.339706

Table 3 - LD for rsl208285

6 134200493 rsl208285 6 134192149 rs9389106 1

6 134200493 rsl208285 6 134200493 rsl208285 1

6 134200493 rsl208285 6 134240872 rs9375991 0.713331

6 134200493 rsl208285 6 134231592 rs9402538 0.685575

6 134200493 rsl208285 6 134233849 rs9321421 0.685575

6 134200493 rsl208285 6 134235209 rsl7062983 0.685575

6 134200493 rsl208285 6 134235552 rsl001333 0.685575 6 134200493 rsl208285 6 134239731 rs9375990 0.685575

6 134200493 rsl208285 6 134241723 rs9402540 0.685575

6 134200493 rsl208285 6 134220098 rs9375987 0.684464

6 134200493 rsl208285 6 134242202 rs2184717 0.684464

6 134200493 rsl208285 6 134211817 rs9375986 0.679317

6 134200493 rsl208285 6 134246785 rs969282 0.656108

6 134200493 rsl208285 6 134212045 rs6903612 0.651746

6 134200493 rsl208285 6 134245038 rs9375992 0.647347

6 134200493 rsl208285 6 134213172 rsl029212 0.64427

6 134200493 rsl208285 6 134212242 rs9389108 0.64324

6 134200493 rsl208285 6 134243782 rs9402543 0.641595

6 134200493 rsl208285 6 134214643 rsll26392 0.638759

6 134200493 rsl208285 6 134247534 rs6919980 0.617673

Table 4 - LD for rs2228671

19 11071912 rs2228671 19 11063306 rs6511720 0.640971

19 11071912 rs2228671 19 11067530 rs8110695 0.609764

19 11071912 rs2228671 19 11056030 rsl 1668477 0.383424

19 11071912 rs2228671 19 11047711 rs9305020 0.355359

19 11071912 rs2228671 19 10721215 rsl965767 0.331887

19* 11071912 rs2228671 19 10702472 rsl 529744 > 0.32832

19 11071912 rs2228671 19 10717418 rs7247567 0.275841

19 11071912 rs2228671 19 10664395 rs2010492 0.261446

19 11071912 rs2228671 19 10639647 rs8099926 0.259328

19 11071912 rs2228671 19 10651143 rs2738042 0.257287

19 11071912 rs2228671 19 10654749 rs2738039 0.257287

19 11071912 rs2228671 19 10662185 rs8102380 0.257287

19 11071912 rs2228671 19 10669770 rs4804519 0.257287

19 11071912 rs2228671 19 10728219 rsl0775614 0.257129

19 11071912 rs2228671 19 10651162 rs2569512 0.255262

19 11071912 rs2228671 19 10615905 rsl0948 0.252224

19 11071912 rs2228671 19 10613968 rsl0405617 0.251383

19 11071912 rs2228671 19 10649813 rs6511708 0.251383

19 11071912 rs2228671 19 10764972 rsll880613 0.247573

19 11071912 rs2228671 19 10770953 rsll881315 0.247573 Table 5 - LD for rsll216185

11 116288184 rsll216185 1 1 116252492 rsl7120119

11 116288184 rsll216185 1 1 116253431 rsl2279433

11 116288184 rsll216185 1 1 116253954 rsl2286581

11 116288184 rsll216185 1 1 116258762 rsl2275565

11 116288184 rsll216185 1 1 116259475 rsl2416987

11 116288184 rsll216185 1 1 116261393 rs2044426

11 116288184 rsll216185 1 1 116262196 rsl0892040

11 116288184 rsll216185 1 1 116262458 rsll216174

11 116288184 rsll216185 1 1 116262608 rsll216175

11 116288184 rsl 1216185 1 1 116263003 rsl2284346

11 116288184 rsl 1216185 1 1 116263903 rsl2292278

11 116288184 rsl1216185 1 1 116265034 rsl2294191

11 116288184 rsll216185 1 1 116277696 rsl7120131

11 116288184 rsll216185 1 1 116278528 rsl7120132

11 116288184 rsll216185 1 1 116278971 rsl7120136

11 116288184 rsll216185 1 1 116280406 rsl2280030

11 116288184 rsll216185 1 1 116288184 rsll216185

11 116288184 rsll216185 1 1 116289902 rsll216186

11 116288184 rsll216185 1 1 116293343 rsl006176

11 116288184 rsll216185 1 1 116317838 rsl7120157

Table 6 - LD for rsl1712066

3 153312999 rsll712066 3 153267641 rs4679963 1

3 153312999 rsll712066 3 153270383 rsl3078000 1

3 153312999 rsll712066 3 153270580 rsl7204830 1

3 153312999 rsll712066 3 153271222 rs4679967 1

3 153312999 rsll712066 3 153273035 rs4679970 1

3 153312999 rsll712066 3 153273377 rs4679971 1

3 153312999 rsll712066 3 153275397 rs4679973 1

3 153312999 rsll712066 3 153275672 rs4679974 1

3 153312999 rsll712066 3 153276362 rsl3094219 1

3 153312999 rsll712066 3 153276605 rsl7283374 1

3 153312999 rsll712066 3 153277806 rsl7283381 1

3 153312999 rsll712066 3 153277946 rsl7283395 1 3 153312999 rsll712066 3 153278274 rsl7204851

3 153312999 rsll712066 3 153278710 rsl7204858

3 153312999 rsll712066 3 153279417 rsl7204865

3 153312999 rsll712066 3 153281349 rsl3069494

3 153312999 rsll712066 3 153282429 rsl3098573

3 153312999 rsll712066 3 153282552 rsl3060417

3 153312999 rsl1712066 3 153282707 rsl3080568

3 153312999 rsll712066 3 153282930 rsl3081237 Table 7 - LD for rsl2721025

11 116706047 rsl2721025 11 116705719 rsl2718462 0.955952

11 116706047 rsl2721026 11 116707401 rs12718464 0.918973

11 116706047 rsl2721027 11 116715784 rsl42174850 0.901494

11 116706047 rsl2721028 11 116735460 rsl7120099 0.897603

11 116706047 rsl2721029 11 116726709 rsll2618266 0.897377

11 116706047 rsl2721030 11 116749072 rs75294699 0.894855

11 116706047 rsl2721031 11 116747282 rsl7120119 0.894242

11 116706047 rsl2721032 11 116748169 rsl2278117 0.894045

11 116706047 rsl2721033 11 116748221 rsl2279433 0.894031

11 116706047 rsl2721034 11 116748515 rsl2292614 0.893971

11 116706047 rsl2721035 11 116748744 rsl2286581 0.893902

11 116706047 rsl2721036 11 116754265 rsl2416987 0.893017

11 116706047 rsl2721037 11 116756183 rs2044426 0.892939

11 116706047 rsl2721038 11 116756986 rsl0892040 0.892907

11 116706047 rsl2721039 11 116757248 rsll216174 0.892893

11 116706047 rsl2721040 11 116757398 rsll216175 0.892887

11 116706047 rsl2721041 11 116757793 rsl2284346 0.892887

11 116706047 rsl2721042 11 116753552 rsl2275565 0.892737

11 116706047 rsl2721043 11 116759824 rsl2294191 0.892475

11 116706047 rsl2721044 11 116762553 rsl44010715 0.892154

11 116706047 rsl2721045 11 116758693 rsl2292278 0.891648 Table 8. Relative risks for IHPS in infants according to total cholesterol levels in plasma from umbilical cord blood

Case Control

RR (95% CI) Subjects Subjects

n Mean n Mean

All 46 1.69 189 1.95 0.02"

Quartile 1 (cholesterol level≤ 1.4999)° 19 1.23 40 1.29 1.00 (reference)

Quartile 2 (1.4999 < cholesterol level≤ 1.8102) 10 1.63 49 1.65 0.47 (0.19-1.13)

Quartile 3 (1.8102 < cholesterol level≤ 2.1722) 11 1.95 47 1.99 0.46 (0.19-1.11)

Quartile 4 (2.1722 < cholesterol level) 6 2.74 53 2.67 0.21 (0.08-0.61 )

RR per mmol/L - - - - 0.36 (0.17-0.74) 0.005° a:Quartiles of total cholesterol (mmol/L) with 25% of the observations in each category. B:Test for no difference in risks between quartiles.

OTest for no change in risk per mmol/L.

Table 9. Relative risks (RRs) for IHPS in infants according to LDL and HDL cholesterol and triglyceride levels in plasma from umbilical cord blood

n Mean n Mean RR (95% CI) P

LDL cholesterol 46 0.60 189 0.68 0.13 ^d

Quartile 1 (LDL level≤ 0.4655)^a 15 0.34 44 0.36 1.00 (reference)

Quartile 2 (0.4655 < LDL level≤ 0.6206) 15 0.54 44 0.54 0.95 (0.41-2.21)

Quartile 3 (0.6206 < LDL level is 0.8275) 9 0.73 49 0.70 0.50 (0.20-1.29)

Quartile 4 (0.8275 < LDL level) 7 1.12 52 1.05 0.37 (0.13-1.01)

RR per mmol/L - - - - 0.32 (0.09-1.14) 0.08°

HDL cholesterol 46 0.49 189 0.56 0.09 "

Quartile 1 (HDL level≤ 0.3620) 17 0.26 42 0.30 1.00 (reference)

Quartile 2 (0.3620 < HDL level≤ 0.5172) 12 0.44 47 0.44 0.61 (0.26-1.41)

Quartile 3 (0.5172 < HDL level≤ 0.6724) 7 0.59 51 0.59 0.30 (0.11-0.82)

Quartile 4 (0.6724 < HDL level) 10 0.89 49 0.86 0.45 (0.18-1.14)

RR per mmol/L - - - - 0.22 (0.05-1.09)

Triglycerides 46 0.41 189 0.45

Quartile 1 (triglyceride level≤ 0.3156) 21 0.23 38 0.26 1.00 (reference)

Quartile 2 (0.3156 < triglyceride level≤ 0.4057) 8 0.37 51 0.37 0.24 (0.09-0.63)

Quartile 3 (0.4057 < triglyceride level≤ 0.4959) 7 0.45 51 0.45 0.22 (0.08-0.59)

Quartile 4 (0.4959 < triglyceride level) 10 0.79 49 0.70 0.36 (0.15-0.88)

RR per mmol/L - - - - 0.31 (0.05-1.91) ^aQuartiles of LDL cholesterol, HDL cholesterol, or triglycerides (mmol/L) with 25% of the observations in each category. "Test for no difference in risks between quartiles.

'Test for no change in risk per mmol/L. Reference List

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Claims

1. An in vitro method for diagnosing a genetic predisposition or susceptibility for pyloric stenosis in a mammal, comprising detecting in a sample obtained from said mammal at least one single nucleotide polymorphism (SNP) in a nucleic acid or fragment thereof, wherein said at least one SNP is selected from the group consisting of the SNPs rsll712066, rs29784, rs573872, rsll216185, rsl208285, rs2228671, rsl2721025 and SNPs in linkage disequilibrium therewith, whereby at least one allele-specific haplotype is determined.

2. The method according to claim 1, further comprising the steps of determining the expression level of at least one of the genes selected from the group consisting of MBNL1, NKX2-5, C3orf79, SGEF, P2RY1, RAP2B, BNIP1, MGC34034, TCF21, AADAC, SUCNR1, SI 3, APOA1, APOC3, APOA4, APOA5 and LDLR, DHX36, GPR149, TMEM14E, C5orf41, ATP6VOE1, SNORA74B, PAFAH1B2, SIDT2, TAGLN, PCSK7 and LOC401093, comparing said expression level to a predetermined expression level, and determining a genetic predisposition or susceptibility for pyloric stenosis for said mammal if said at least one allele-specific haplotype is determined and said expression level is significantly different from said

predetermined expression level.

3. The method according to any of claims 1-2, wherein said at least one SNP is one SNP, such as 2 SNPs, such as 3 SNPs, such as 4 SNPs, such as 5 SNPs, such as 7 SNPs.

4. The method according to any of claims 1-3, wherein said SNPs in linkage disequilibrium is set forth in table 1-7.

5. The method according to any of claims 2-4, wherein said at least one gene is one gene, such as 2 genes, such as 4 genes, such as 6 genes, such as 8 genes, such as 10 genes, such as 12 genes, such as 14 genes, such as 16 genes, such as 18 genes, such as 20 genes.

6. The method according to any of claims 1-5, wherein the mammal is a human infant.

7. The method according to claim 6, wherein said human infant is selected from the group consisting of a boy and a girl.

8. The method according to claim 7, wherein said infant is less than 2 months old, such as less than 6 weeks old, such as less than 4 weeks old, such as less than 2 weeks old, such as less than 1 week old, such as less than 4 days old, such as less than 2 days old, such as less than one day old.

9. The method according to any of claims 1-8, wherein said nucleic acid is a DNA, genomic DNA, RNA, cDNA, hnRNA and/or mRNA.

10. The method according to any of claims 1-9, wherein said detection is accomplished by sequencing, mini-sequencing, hybridization, restriction fragment analysis, oligonucleotide ligation assay, allele specific PCR.

11. The method according to claim 2, wherein the predetermined expression level is the expression level from an infant not suffering from pyloric stenosis.

12. The method according to any of claims 1-11, wherein the comparison of expression levels is done at a predefined statistical significance.

13. A diagnostic kit and/or a research kit, comprising at least one combination of probes for detecting at least one of the haplotypes of claim 1 and/or probes for determining the expression level the genes of claim 2.

14. Use of cholesterol for preparing a medicament for the treatment of pyloric stenosis in infants.