The Genetics of CCHS

by Melinda Riccitelli, CCHS mom and professor of Biology at Mira Costa College, CA

In March of 2003, the Necker Research team, (France) established that mutation of the PHOX2B gene was the causative agent of CCHS. Mutations can follow a polyalanine expansion mutation (PARM) or non-polyalanine expansion mutation pattern (non-PARM). With the development of a mouse model much is being learned about this very rare disease. To comprehend the genetic basis of CCHS, a basic understanding of genetics is needed.

What is Genetics?

Genetics is the study of genes and inheritance patterns. As a field of science it is relatively new. The father of modern genetics is Greger Mendel, a monk who lived in the 1850’s. His experiments with plant mating, worked out that traits were inherited discreetly in the form of a gene. Each trait is controlled by two genes, but each parent can give only one gene to an offspring. Mendel’s work was lost to the scientific community until the early 1900’s when chromosomes and cellular division were discovered. It still took 50 years for DNA to be identified as the genetic material. Hershey and Chase definitively showed, in one of the most elegant experiments of the twentieth century, DNA was the genetic material. A few years later, Watson and Crick discovered the structure of DNA. With this discovery, the functioning of DNA was realized. DNA controls inheritance through self replication which allows daughter cells to inherit genetic material from a parent cell. DNA controls cellular activity (metabolism) through the production of proteins. Proteins are cellular molecules that do all the work in the cell. Self-replication and protein production is the Central Dogma of modern genetics. Because of these discoveries we know live in an age of genetic manipulation never seen before called the Genomic Age.

What is a Gene?

A gene is a small part of DNA. Genes only make up a VERY small portion of all the DNA in the cell, roughly about 1% of DNA is gene material. Only sequences that code for proteins (or RNA products) are considered genes. Human genes are split between coding and noncoding regions. Only coding regions make up a protein. DNA along with proteins is contained in the nucleus of a cell in a structure called a chromosome. DNA is made up of chemical letters called A (adenine), T (thymine), G (guanine), and C (cytosine). These letters are arranged in a specific sequence and direct the lay-out (sequence) of a protein.

The cell works by converting a specific DNA sequence into a specific sequence of amino acids, the building blocks of proteins. This process occurs across two stages, called transcription (DNA becomes a message) and translation (the message becomes a protein). Genes are actually made up of coding and non-coding regions. Coding regions get transcribed and translated into proteins.

A person’s genetic make-up is described as the genotype. The physical trait (eye color, dimples, freckles …) that the genotype creates is called a person’s phenotype. In CCHS: the genotype is the PHOX2B gene (normal is 20/20–both genes have a very specific 20 sequence alanine repeat in the sequence–I will talk more about this later) and the phenotype is “normal” autonomic nervous system (ANS) (the PHOX2B gene controls development of the ANS).

What is a Genetic Disease?

A genetic disorder is a disease caused by abnormalities in an individual’s genetic material (genome). Genetic disorders can be classified as (1) single-gene, (2) multifactorial, (3) chromosomal, and (4) mitochondrial. Single genes are inherited in recognizable patterns: autosomal dominant, autosomal recessive, and X-linked for example. Genes can be changed in several ways: base substitutions (changing one DNA base for another–these are called missense or nonsense mutations), extensions (part of the gene is repeated), or deletions (part of the gene is missing). Some extension and deletion mutations are called frameshift mutations if the genes reading frame is altered. Changes in PHOX2B are single gene mutations and either classified as PARM or Non-PARM (NPARM) mutations. PARM mutations are extension mutations (patient gets extra poly-Alanine codes); NPARM can be base substitution mutations (missense and nonsense) or deletion mutations.

In multifactorial genetic diseases both the environment and changes in multiple genes determine the disease (heart disease, Alzheimer’s, and cancer are examples). Chromosomal diseases result from gross changes to chromosome structure (Down syndrome is an example of a chromosomal disease). Mitochondrial diseases are rare and due to non-nuclear DNA.

Genetic Research

When researchers study a disease they hope to identify a gene associated with the condition. With this information they try to understand the normal functioning of the gene and how alterations change this function. Identification and understanding of normal verses abnormal function can lead to improved diagnosis and treatment options:

1. Prenatal diagnosis: testing for a hereditary disease during pregnancy

2. Pre-implantation diagnosis: testing a fertilized egg for disease, followed by selection of healthy embryo for in vitro fertilization procedures.

3. Screening for people at risk: testing for disease onset before clinical symptoms becomes obvious.

4. Developing new treatments: identifying the gene (or protein) involved in a disease makes possible the designing of drugs or treatments that counter the defects of the gene. In addition, possible genetic therapy protocols may become visible once the gene is identified (gene therapy, in its infancy, has the goal to replace damaged genes with normal ones to permanently eliminate the condition).

The PHOX2B Gene


The PHOX2B gene has a role in autonomic nervous system development (ANS). The ANS is part of the Peripheral Nervous System (PNS) that carries responses out of the brain to various muscles and glands in the body. The ANS regulates involuntary and natural behaviors like breathing, heart rate, blood glucose levels, and body temperature.

The PHOX2B gene is a homeotic, or master control gene. These genes are important in fetal development because they regulate other genes. In fact it is known that the PHOX2B protein regulates other ANS genes (PHOX2A, dopamine-β-hydroxylase, and TLX-2 have been identified at this time) by binding to their activation site (a region on a gene called the promoter). This binding tells these genes what to do. All of these genes need to be expressed (activated) properly to develop full/normal autonomic responses. A mutated PHOX2B gene results in several changes in the protein’s function: transcription of these other genes is disrupted, DNA binding to these genes is reduced, mutated proteins clump (aggregate) together in the cytoplasm of the cell, and mutated proteins are relocated out of the nucleus and into the cytoplasm. These changes are thought to be responsible for the CCHS phenotype (a compromised respiratory drive, irregular heart rate, unable to control body temperature…).

Although most original CCHS patients are a product of a de novo mutation (brand new), once present the mutation inherits as an autosomal dominant. A CCHS patient, therefore, has a 50% chance of passing the defect onto offspring. A diagram of transmission is seen in Figure 1.


The difference between PARM and NPARM is the kind of mutation the gene has. Remember a mutation just means the DNA has CHANGED. PARM and NPARM describes HOW it has changed. PARM stands for Poly Alanine Repeat Mutation. Alanine is an amino acid found in proteins (remember proteins do all the work in the cell; DNA decides what protein is going to get made in the cell).

In a “normal” PHOX2B protein there are 20 alanines in a row. To get an alanine in the protein the gene has to code for it. Genes code with the letters A,T,G,C in a particular sequence. The amino acid alanine is actually coded for by 4 distinct DNA sequences–CGA, CGG, CGT, or CGC–ANYTIME these sequences are seen in the gene, alanine will be placed in the protein by the cellular machinery that makes proteins. So, if the DNA has one (1) CGA sequence -> the protein it makes gets 1 alanine; 2 CGA -> 2 alanines; 20 CGA -> 20 alanines.

PHOX2B PARM mutations occur in the third coding region of the gene. In this region there is a sequence of bases that code for 20 alanines in the protein. With a mutated PHOX2B you start to get additional DNA sequences that code for alanine. A person, then, with a 20/25 has one chromosome (DNA) with 20 CGA sequences that puts 20 alanines in a protein that is normal -> nothing is wrong with this protein; on the other chromosome of this person (remember everyone has 2 chromosomes or genes for each trait) they have 25 CGA sequences that puts 25 alanines in the protein -> this protein does not work the way it is supposed to.

NPARM just means that the PHOX2B mutation is not a result of additional alanine sequences (think CGA) causing the problem. In NPARM the gene is changed in some other way in different coding regions of PHOX2B. Perhaps there was a single base change (base substitutions) in a DNA sequence that changed the amino acid in the protein, damaging the protein, altering its cellular function. These mutations are much harder to detect – because small changes are being made. These mutations are detected through DNA sequence analysis – you have to isolate the DNA and sequence it base, by base, by base, by base… until you see what is wrong. PARM mutations are easier to detect because as the gene expands with more alanine sequences, the DNA gets BIGGER by many sequences (an expansion to 25 alanines in the protein would add 15 bases to the gene). DNA can easily be separated by size–a 20 gene will separate from a 25 gene on a gel–easy to see, easy to pick up.

Some NPARM mutations are caused by multiple base-pair deletions that cause a frameshift in the reading frame of the gene. These mutations generally produce severe disease.

Because of the reduction in price of gene sequencing, most genetic test centers now sequence the PHOX2b gene to determine if a patient has CCHS.

CCHS Genotype/CCHS Phenotype

It is generally believed that that more alanine expansion the patient has, the more complex the CCHS phenotype is. NPARMs are generally seen as more severely affected than PARMs. But each CCHS patient is unique: there are 20/27 patients that are vent dependent 24 hours a day/seven days a week, while other 20/27 patients are night-only vent dependent. The same is true for NPARMs, while many are complex some are very mild. The CCHS phenotype is very variable. Each case then has to be viewed and treated based on symptoms presented by the patient, as well as considering the mutation number and type to give clues as to how the condition may present. To rely simply on the mutation number or type as an “absolute” description of phenotype is ill-advised.

CCHS Mosaics

Mosaic is a difficult term to define and understand. A human body is made up of TRILLIONS of cells. When we talk about a person’s genotype (the genes a person has)–we would say that for most people all these cells have the SAME genetic information in them (this is a very simplistic way of looking at it). Therefore, in a “normal” person–the genotype is identical in each cell.

A mosaic individual has cells with different DNA information in them–all the cells are not the same. In a CCHS mosaics – some of the cells will be 20/20 others will be 20/27. Because the change is not in every cell it is hard to find them. That is why the specialist says that there is a 50/50 chance of finding the “unique” cell(s) in a 100 test sample. How many cells are damaged depends on when, post-zygotically, the mutation occurred.

Again, let’s look at it in a simple way. If we are looking at a PARM mutation occurring de novo, pre-zygotically (before fertilization of the egg by the sperm) when that fertilization occurs and the zygote forms (one big cell) the genotype is established in all cells. That one cell starts to divide into 2, 4, 8, 16, 32… ball of cells -> all identical. Fetus develops, all the cells have the PARM mutation -> child born with CCHS.

Mosaics pre-zygotically have 2 normal PHOX2B genes. Fertilization occurs and the zygote is “normal” for PHOX2B. The zygote starts to divide 2, 4, 8, 16, 32… in one of these cells PHOX2B mutates after the zygote has formed (mosaicism due to POST zygotic mutation) all cells formed from this cell line will be damaged. All cells formed from other zygotic cells are normal. So a CCHS mosaic has some cells 20/20 and some cells 20/27. Since not every cell is abnormal it is harder to find them by testing (although it can be done). Mosaics can also be asymptomatic or have mild CCHS disease. But, they can also pass the CCHS genotype to offspring. The transmission, however, does not follow a typical dominant transmission pattern. Oftentimes mosaic parents are diagnosed after the birth of a CCHS child.

Genetic Testing of CCHS

A CCHS diagnosis can now be validated with genetic testing. There are several genetic testing labs in the United States (link to testing centers in resource section). Many labs can also now assist in prenatal/preimplantation screening.